Heterodimers of IL-15 and IL-15R alpha to increase thymic output and to treat lymphopenia

ABSTRACT

The present invention provides method for promoting the maturation and export of T cells from thymic tissue by contacting the thymic tissue with supraphysiological levels of interleukin (IL)-15. The present invention also provides methods for preventing, alleviating, reducing, and/or inhibiting lymphopenia or peripheral depletion of lymphocytes in a patient in need thereof by administering to the patient IL-15.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/390,504, filed Feb. 14, 2012, U.S. Pat. No. 8,871,191; which is aU.S. National Stage Application of International Application No.PCT/US2010/045511, filed Aug. 13, 2010, which claims the benefit of U.S.provisional application No. 61/234,152 , filed on Aug. 14, 2009; andU.S. provisional application No. 61/234,155, filed Aug. 14, 2009. Eachapplication is herein incorporated by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TXT FILE

This application includes a Sequence Listing as a text file named“77867-921971-SEQLIST.txt” created Oct. 10, 2014 and containing 65,820bytes. The material contained in this text file is incorporated byreference.

FIELD OF THE INVENTION

The present invention provides compositions and methods for promotingthe maturation and export of T cells from the thymus, e.g., toperipheral lymphoid and non-lymphoid tissues by contacting the thymustissue, in vitro or in vivo, with interleukin (IL)-15.

The invention additionally provides methods for preventing, alleviating,reducing, and/or inhibiting lymphopenia or depletion of lymphocytes inperipheral tissues in a patient in need thereof by administering IL-15to the patient.

BACKGROUND OF THE INVENTION

Two common gamma-chain cytokines, IL-2 and IL-7 are currently approvedor considered for both AIDS and cancer immunotherapy. See, Sportes, etal., (2008) J Exp Med 205:1701-1714; Levy, Y. (2009) J Clin Invest.119(4):997-100785; and Rosenberg, et al., (2006) J Immunother29:313-319. No clinical experience exists with the gamma-chain cytokineIL-15. See, Cheever, (2008) Immunological Reviews 222:357-368.

IL-15 is a non-redundant cytokine important for the development,survival, and proliferation of natural killer (NK) and CD8+ T-cells. Itshares with IL-2 the same IL-2 beta gamma receptor and has many similareffects on lymphocytes, but unlike IL-2 is not produced by lymphocytesbut by a plethora of other cells including, importantly, antigenpresenting cells and macrophages, and stroma cells in several tissues.The biological effects of IL-2 and IL-15 at the level of the organismare dramatically different, as shown by work in knockout mice: lack ofIL-15 causes immune system defects, whereas lack of IL-2 causes immuneactivation and severe autoimmunity. See, Waldmann, (2006) Nat RevImmunol 6:595-601; and Ma, et al., (2006) Annu Rev Immunol 24:657-679.Both cytokines are under tight and complex regulation at all steps ofexpression and secretion. The biological differences of IL-2 and IL-15are determined by their different production sites, their strength ofassociation with membrane receptor proteins termed IL-2 Receptor alphaand IL-15 Receptor alpha (IL-15Rα), respectively, and the regulation ofthese extra receptor molecules. IL-15 has been also reported to have aunique mechanism of action in vivo among the common gamma chaincytokines: IL-15 functions in a complex with IL-15Rα and depends on theco-expression by the same cells of IL-15Rα. See, Burkett, et al., (2004)J Exp Med 200:825-834; Burkett, et al., (2003) Proc Natl Acad Sci USA100:4724-4729; Dubois, et al., (2002) Immunity 17:537-547; Sandau, etal, (2004) J Immunol 173:6537-6541; Schluns, et al., (2004) Blood103:988-994; Rubinstein, et al., (2006) Proc Natl Acad Sci USA103:9166-9171; Bergamaschi, et al., (2008) J Biol Chem 283:4189-4199.IL-15 has non-redundant roles in the development and function of NK andintestinal intraepithelial lymphocytes (IELs). See, Cooper, et al.,(2001) Blood 97:3146-3151. It stimulates cytolytic activity, cytokinesecretion, proliferation and survival of NK cells. See, Fehniger, etal., (1999) J Immunol 162:4511-4520; Ross, et al., (1997) Blood89:910-918; and Carson, et al., (1994) J Exp Med 180:1395-1403. IL-15has a proliferative and survival effect on CD8+ memory T-cells and naiveCD8+ T-cells. See, Tan, et al., (2002) J Exp Med 195:1523-1532; Zhang,et al., (1998) Immunity 8:591-599; Berard, et al., (2003) J Immunol170:5018-5026; and Alves, et al., (2003) Blood 102:2541-2546.

Several studies have evaluated the effects of IL-15 administration invivo. CD8+ memory T-cell proliferation increased after a single dose ofIL-15 in normal mice. See, Zhang, et al., (1998) Immunity 8:591-599.Administration of IL-15 to mice enhanced the antitumor activity aftersyngeneic bone marrow transplantation (BMT) and antigen-specific primaryCD8+ T-cell responses following vaccination with peptide-pulseddendritic cells. See, Rubinstein, et al., (2002) J Immunol169:4928-4935; Katsanis, et al., (1996) Transplantation 62:872-875.IL-15 also enhanced immune reconstitution after allogeneic bone marrowtransplantation. See, Alpdogan, et al., (2005) Blood 105:865-873; andEvans, et al., (1997) Cell Immunol 179:66-73. The ability of IL-15 topromote growth, survival and activation of key lymphocyte populationsmake it also an attractive candidate for supporting growth in vitro andin vivo of cells for adoptive cell therapy. See, Rosenberg, et al.,(2008) Nat Rev Cancer 8:299-308; and Berger, et al., (2008) J ClinInvest 118:294-305.

We have demonstrated that efficient production of IL-15 requires theexpression of IL-15 and IL-15 Receptor alpha (IL-15Rα) in the same cell.See, Bergamaschi, et al., (2008) J Biol Chem 283:4189-4199.Co-production leads to intracellular association of IL-15 and IL-15Rα inthe endoplasmic reticulum, stabilization of both molecules and efficienttransport to the cell surface (FIG. 1). We showed that an additionalcritical step is the rapid cleavage and release of the IL-15/IL-15Rαcomplex from the cell surface, both in vitro and in vivo, resulting in asoluble, systemically active form of IL-15/IL-15Rα, in addition to thebioactive complex on the cell surface. See, Dubois, et al., (2002)Immunity 17:537-547; Bergamaschi, et al., (2008) J Biol Chem283:4189-4199; and Budagian, et al., (2004) J Biol Chem 279:40368-40375.Our experiments using IL-15 complexed to a deletion mutant of IL-15Rαcontaining only the soluble Receptor alpha extracellular fragmentdemonstrated that this complex is bioactive in vivo in the absence ofany membrane-bound form.

Therefore, we proposed that IL-15Rα is part of a heterodimeric IL-15cytokine, rather than functioning as a cytokine receptor. These resultshave been supported by other investigators, and provide the basis for abetter understanding of IL-15 biology. See, Duitman, et al., (2008) MolCell Biol 28:4851-4861; Mortier, et al., (2008) J Exp Med 205:1213-1225.The results also provide the molecular basis to explain some intriguingobservations, including the requirement of production of IL-15 andIL-15Rα from the same cells for appropriate function in vivo. See,Sandau, et al., (2004) J Immunol 173:6537-6541; and Koka, et al., (2003)J Exp Med 197:977-984. Such results are fully explained by our findingthat stabilization during co-expression in the same cell is required forphysiological levels of IL-15 production. It has also been reported thatthe cells that physiologically express IL-15 also express IL-15Rα,consistent with IL-15 production as a heterodimer in the body. See,Dubois, et al., (2002) Immunity 17:537-547; Giri, et al., (1995) JLeukoc Biol 57:763-766; and Ruckert, et al., (2003) Eur J Immunol33:3493-3503. Interpretation of all data available to date suggests thatthe main bioactive form of IL-15 is in a complex with the Receptor alphaeither on the surface of the cells or in a soluble circulating form. Itremains to be determined whether single-chain IL-15 is produced in thebody in physiologically relevant levels and what is its exact function.

It has been previously reported that IL-15 secretion is inefficient.See, Bamford, et al., (1998) J Immunol 160:4418-4426; Gaggero, et al.,(1999) Eur J Immunol 29:1265-1274; Kurys, et al., (2000) J Biol Chem275:30653-30659; Onu, et al., (1997) J Immunol 158:255-262; and Tagaya,et al., (1997) Proc Natl Acad Sci USA 94:14444-14449. We took asystematic approach to develop IL-15 expression vectors producing highlevels of bioactive cytokine based on the observation that multipleregulatory steps during gene expression create bottlenecks of IL-15production. See, Jalah, et al., (2007) DNA Cell Biol 26:827-840; andKutzler, et al., (2005) J Immunol 175:112-123. We showed thatcombination of two approaches, namely mRNA optimization (RNA/codonoptimization) of the IL-15 coding sequences and substitution of thesignal peptide with other efficient secretory signals resulted insynergistically improved expression and secretion of bioactive IL-15.See, Jalah, et al., (2007) DNA Cell Biol 26:827-840. Taking advantage ofthe stabilization of IL-15 by co-expression with IL-15Rα describedabove, we produced equally optimized vectors for IL-15Rα and combinationvectors expressing both molecules, as well as combinations producingonly the soluble heterodimeric cytokine. The final improvement inexpression of secreted IL-15 was more than 1,000 fold compared to wtIL-15 cDNA, as determined by in vitro and in vivo experiments. We haveproduced similar vectors for mouse, macaque and human IL-15/IL-15Rα.

Two forms of interleukin-15 (IL-15) are known, containing a long signalpeptide (LSP) or a short signal peptide (SSP), respectively. The twoforms are produced by alternatively spliced mRNAs and differ only in thelength of their signal peptides, the 48 aa long signal peptide or the 21aa short signal peptide (120, 121, 125-127). See, Onu, et al., (1997) JImmunol 158:255-262; Tagaya, et al., (1997) Proc Natl Acad Sci USA94:14444-14449; Meazza, et al., (1997) Eur J Immunol 27:1049-1054;Meazza, et al., (1996) Oncogene 12:2187-2192; and Nishimura, et al.,(1998) J Immunol 160:936-942. Whereas LSP IL-15 is secreted, SSP IL-15remains exclusively intracellular and its function is not known. It hasbeen proposed that SSP IL-15 may have a regulatory function since it wasdetected both in the cytoplasm and the nucleus of DNA-transfected cells.The SSP signal affects both stability and localization of IL-15, sincelower levels of the SSP isoform were detected when the two isoforms wereexpressed from similar vectors. See, See, Onu, et al., (1997) J Immunol158:255-262; Tagaya, et al., (1997) Proc Natl Acad Sci USA94:14444-14449; and Bergamaschi, et al., (2009) J Immunol, 5:3064-72.

In Bergamaschi, we showed that, similar to LSP IL-15, SSP IL-15 isstabilized and secreted efficiently upon coexpression of IL-15Rα in thesame cell. See, Bergamaschi, et al., (2009) J Immunol, supra.Co-expression of SSP IL-15 and IL-15Rα in mice showed increased plasmalevels of bioactive SSP IL-15 and mobilization and expansion of NK and Tcells. Therefore, SSP IL-15 is secreted and bioactive when produced as aheterodimer with IL-15Rα in the same cell. The apparent stability ofthis complex both in vitro and in vivo is lower compared to LSPIL-15/IL-15Rα complex, as revealed by direct comparisons. This resultsin lower production of secreted bioactive IL-15/IL-15Rα. Thus,alternative splicing may provide the cell with the ability to producedifferent levels of bioactive IL-15. Since both forms of IL-15 may beproduced in the same cell by alternative splicing, an additional levelof regulation is possible. We showed that when both LSP IL-15 and SSPIL-15 are produced in the same cell they compete for the binding toIL-15Rα, resulting in lower levels of bioactive IL-15. Therefore,co-expressed SSP IL-15 acts as competitive inhibitor of LSP IL-15. Thissuggests that usage of alternative splicing is an additional level ofcontrol of IL-15 activity. Expression of both SSP and LSP forms of IL-15appears to be conserved in many mammals, suggesting that SSP may beimportant for expressing a form of IL-15 with lower magnitude andduration of biological effects. The present invention is based, in part,on the discovery that SSP IL-15, which is produced in the thymus, isimportant for intrathymic effects on lymphocyte differentiation andmaturation.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions and methods that promote thematuration of T cells in the thymus and the output or migration ofmature and/or activated lymphocytes from a central lymphoid organ toperipheral tissues by administration of IL-15. The invention is based,in part, on the discovery that IL-15 promotes the migration of T cellsout of the thymus and subsequently to peripheral lymphoid (e.g., spleenand lymph node) and non-lymphoid tissues (e.g., lung and liver). In someembodiments, the methods concurrently promote the maturation oflymphocytes in the bone marrow, e.g., B cells and natural killer (NK)cells, and their migration to peripheral lymphoid and non-lymphoidtissues.

Accordingly, in one aspect, the invention provides methods of promotingT-cell maturation in thymic tissue comprising contacting the thymictissue with IL-15.

The thymic tissue can be in vivo or in vitro.

In a related aspect, the invention provides methods of promoting themigration of lymphocytes from a central lymphoid tissue to one or moreperipheral tissues in a subject in need thereof comprising administeringto the subject IL-15.

With respect to the embodiments, in some embodiments, the lymphocytesare T cells and the central lymphoid tissue is thymus. In someembodiments, the lymphocytes are B cells and/or NK cells and the centrallymphoid tissue is bone marrow.

In some embodiments, the lymphocytes migrating from the central lymphoidtissues are mature but not activated. In some embodiments, thelymphocytes migrating from the central lymphoid tissues are mature andactivated. In some embodiments, the T cells migrating from the thymusare mature single positive (CD4+ or CD8+) T cells. The T cells inducedto leave the thymus may be activated or not activated.

The invention additionally provides methods for preventing, treating,alleviating, reducing and/or inhibiting lymphopenia or depletion oflymphocytes in peripheral tissues by administration of IL-15. Thepresent invention further provides methods for promoting therepopulation of peripheral tissues that have been depleted oflymphocytes and accelerating the recovery from lymphocyte depletion ofperipheral tissues by the administration of IL-15.

Accordingly, in one aspect, the invention provides methods ofpreventing, reducing or inhibiting lymphopenia or depletion oflymphocytes in peripheral tissues in an individual in need thereofcomprising systemically administering IL-15 to the individual.

In some embodiments, the lymphopenia or lymphocyte depletion ofperipheral tissues is drug-induced. For example, the individual may bereceiving anticancer drugs or antiviral drugs, or radiation therapy thatinduces lymphopenia or lymphocyte depletion of peripheral tissues.

In some embodiments, the IL-15 is co-administered with an agent thatcauses depletion of lymphocytes in peripheral tissues, e.g., ananticancer or an antiviral agent. In some embodiments, the IL-15 isco-administered with radiation therapy.

In a related aspect, the invention provides methods of promoting oraccelerating the repopulation of lymphocytes in peripheral tissues in anindividual in need thereof comprising systemically administering IL-15to the individual.

In some embodiments, the systemic administration of IL-15 prevents orreduces the depletion of or promotes or accelerates the repopulation ofone or more of T cells, B cells or NK cells. In some embodiments, thesystemic administration of IL-15 prevents or reduces the depletion of orpromotes or accelerates the repopulation of one or more of CD4+ T cellsor CD8+ T cells.

In some embodiments of the methods of the invention, the subject orpatient is a mammal. In some embodiments, the subject or patient is ahuman.

When administered in vivo the IL-15 can be administered systemically,including without limitation, enterally (i.e., orally) or parenterally,e.g., intravenously, intramuscularly, subcutaneously, intradermally,intranasally, or inhalationally. In some embodiments, the IL-15 isadministered locally, for example, intrathymically.

Systemic administration is at a dose that is sufficient to maintainIL-15 at supraphysiologic levels. For example, IL-15 DNA or protein canbe administered at a dose sufficient to achieve plasma levels of IL-15of about 1 to 1000 ng/ml, for example, plasma levels of IL-15 of about10 to 1000 ng/ml. The IL-15 and IL-15Rα can be delivered in equimolaramounts. Such a range of IL-15 plasma concentrations can be achieved,e.g., after intramuscular electroporation of about 0.1 mg IL-15/IL-15Rαexpressing DNA plasmid per kg body weight. Alternatively, anIL-15/IL-15Rα protein complex can be administered at a dose of about0.01 to 0.5 mg/kg. IL-15/IL-15Rα polypeptides can be administered, e.g.,subcutaneously, intramuscularly, intraperitoneally or intravenously.See, e.g., Rosati, et al., Vaccine (2008) 26:5223-5229.

The IL-15 can be administered as a polypeptide or as a polynucleotideencoding IL-15. In some embodiments, the IL-15 is co-administered withIL-15Rα, e.g., as a heterodimer. The co-administered IL-15Rα can be apolypeptide or a polynucleotide encoding IL-15Rα. The co-administeredIL-15Rα can be in the same or different form as the IL-15. For example,both the IL-15 and the IL-15Rα can be administered as polypeptides or asone or more polynucleotides encoding IL-15 and/or IL15Rα. Alternatively,one of the IL-15 and the IL15Rα can be administered as a polypeptide andthe other as a polynucleotide encoding either IL-15 or IL-15Rα. In someembodiments, the IL-15Rα is a soluble IL-15Rα. In some embodiments, theIL-15Rα may be administered in the form of an Fc fusion protein or apolynucleotide that encodes an Fc fusion protein.

In some embodiments, the IL-15 and the IL-15Rα are concurrentlyadministered as one or more polynucleotides encoding IL-15 and/orIL-15Rα. The polynucleotide encoding IL-15 and the polynucleotideencoding IL-15Rα can be on the same or separate vectors, for example,single or multiple plasmid vectors. In some embodiments, the IL-15 andthe IL-15Rα polynucleotides are concurrently expressed from a plasmidvector of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQID NO: 19.

In some embodiments, the polynucleotides encoding one or both of IL-15and the IL-15Rα are wild-type coding sequences. In some embodiments, thepolynucleotide encoding IL-15 shares at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1. Insome embodiments, the polynucleotide encoding IL-15Rα shares at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO:5 or SEQ ID NO:7.

In some embodiments, the polynucleotides encoding one or both of IL-15and the IL-15Rα are codon optimized for improved expression over thewild-type coding sequences. In some embodiments, the polynucleotideencoding IL-15 shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to SEQ ID NO:3 or SEQ ID NO:4. Insome embodiments, the polynucleotide encoding IL-15Rα shares at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99° A or 100% sequenceidentity to SEQ ID NO:9 or SEQ ID NO:11.

When expressed from a polynucleotide encoding IL-15, the coding sequencecan have a native or a heterologous signal peptide. In some embodiments,the signal peptide is a native IL-15 signal peptide, for example, thenative IL-15 long signal peptide or the native IL-15 short signalpeptide. In some embodiments, the signal peptide is a heterologoussignal peptide, for example, a signal peptide fromgranulocyte-macrophage colony stimulating factor (GM-CSF), tissueplasminogen activator (tPA), growth hormone, or an immunoglobulin.

In some embodiments, the peripheral tissue is a peripheral lymphoidtissue, including without limitation, spleen, lymph node,mucosal-associated lymphoid tissues (MALT), e.g., tonsils and/orgut-associated lymphoid tissues (GALT), including Peyer's patches.

In some embodiments, the peripheral tissue is a peripheral non-lymphoidtissue, e.g., lung, liver, kidney, heart, skin, etc.

Preferably, the IL-15 is administered without an antigen, i.e., is notco-administered with an antigen.

In a related aspect, the invention provides a DNA vector encoding IL-15and IL-15Rα for use in promoting lymphocyte mobilization from centrallymphoid tissue and migration to peripheral tissues.

In another aspect, the invention provides IL-15/IL-15Rα for use inpromoting lymphocyte mobilization from central lymphoid tissue andmigration to peripheral tissues.

In a related aspect, the invention provides a DNA vector encoding IL-15and IL-15Rα for use in promoting the maturation and export of T cellsfrom the thymus to peripheral tissues, including peripheral lymphoid andnon-lymphoid tissues.

In another aspect, the invention provides IL-15/IL-15Rα polypeptidecomplexes for use in promoting the maturation and export of T cells fromthe thymus to peripheral tissues, including peripheral lymphoid andnon-lymphoid tissues.

In a related aspect, the invention provides a DNA vector encoding IL-15and IL-15Rα for use in promoting repopulation of depleted lymphocytes inperipheral tissues and/or preventing, reducing and/or inhibitinglymphopenia.

In another aspect, the invention provides IL-15/IL-15Rα polypeptidecomplexes for use in promoting repopulation of depleted lymphocytes inperipheral tissues and/or preventing, reducing and/or inhibitinglymphopenia.

In another aspect, the invention provides stable cell lines that expressIL-15/IL-15Rα polypeptides. In some embodiments, the stable cell lineexpresses IL-15/IL-15Rα in the form of a fusion protein. In someembodiments, the stable cell lines produce IL-15 and IL-15Rα asdifferent molecules. In some embodiments, the stable cell lines produceIL-15 and secreted IL-15Rα deletions that lack the transmembrane anchorportion of the receptor. In some embodiments the stable cell linesproduce IL-15 and fusions of IL15Rα to the an immunoglobulin Fc region.In some embodiments the stable cell lines produce IL-15 and IL-15Rαfusions to polypeptides able to direct binding of the fusion to the cellsurface of specific cell types. In some embodiments the stable celllines produce IL-15 and IL-15Rα fusions to polypeptides able to directmultimerization of the fusion.

Further embodiments are as described herein.

DEFINITIONS

The term “central lymphoid tissue” or “central lymphoid organ” refers tospecialized lymphoid tissues where the production of new lymphocytes, orlymphopoiesis, takes place. For example, T cells develop and mature inthe thymus or thymic tissue. B cells and natural killer (NK) cellsdevelop in bone marrow tissue. See, e.g., Chapter 7 of Janeway, et al.,Immunobiology, 2001, Garland Publishing, New York.

The term “peripheral lymphoid tissue” or “peripheral lymphoid organ”refers to peripheral tissues of highly organized architecture, withdistinct areas of B cells and T cells. Newly produced lymphocytes leavethe central lymphoid tissues, and are carried in the blood to theperipheral lymphoid tissues. Exemplary peripheral lymphoid tissues ororgans include the spleen, lymph nodes, mucosal-associated lymphoidtissues (MALT), e.g., tonsils and gut-associated lymphoid tissues(GALT), including Peyer's patches.

The term “mature lymphocyte” refers to a lymphocyte that is undergoneselection and development to maturity in the central lymphoid tissuesufficient to circulate to peripheral lymphoid tissues. With respect toT cells, a mature T cell is characterized by the expression of eitherCD4 or CD8, but not both (i.e., they are single positive), andexpression of CD3. With respect to B cells, a mature B cell ischaracterized by VDJ rearranged immunoglobulin heavy chain gene, VJrearranged immunoglobulin light chain gene, and the surface expressionof IgD and/or IgM. The mature B cell may also express CD19 and the IL-7receptor on the cell surface.

The term “activated lymphocyte” refers to lymphocytes that haverecognized an antigen bound to a MHC molecule and the simultaneousdelivery of a co-stimulatory signal by a specialized antigen-presentingcell. Activation of lymphocytes changes the expression of severalcell-surface molecules.

With respect to T cells, resting naive T cells express L-selectin, andlow levels of other adhesion molecules such as CD2 and LFA-1. Uponactivation of the T cell, expression of L-selectin is lost and, instead,increased amounts of the integrin VLA-4 are expressed. Activated T cellsalso express higher densities of the adhesion molecules CD2 and LFA-1,increasing the avidity of the interaction of the activated T cell withpotential target cells, and higher densities of the adhesion moleculeCD44. Finally, the isoform of the CD45 molecule expressed by activatedcells changes, by alternative splicing of the RNA transcript of the CD45gene, so that activated T cells express the CD45RO isoform thatassociates with the T-cell receptor and CD4. Also, with respect tocytokine production, resting T cells produce little or no IL-2 and the βand γ subunits of the IL-2 receptor. In contrast, activated T cellsproduce significant amounts IL-2 along with the α chain of the IL-2receptor.

With respect to B cells, activated B cells have undergone isotypeswitching and secrete immunoglobulin. Naive B cells express cell-surfaceIgM and IgD immunoglobulin isotypes. In contrast, activated or memory Bcells express and secrete IgG, IgA or IgE immunoglobulin isotypes.

The terms “output” or “migration” from a central lymphoid tissue refersto migration or export of mature lymphocytes from a central lymphocytetissue to a peripheral tissue, including lymphoid and non-lymphoidperipheral tissues. Output includes the migration of mature T cells fromthe thymus and the migration of mature B cells and NK cells from thebone marrow.

The terms “treating” and “treatment” refer to delaying the onset of,retarding or reversing the progress of, or alleviating or preventingeither the disease or condition to which the term applies, or one ormore symptoms of such disease or condition.

The terms “lymphopenia” or “lymphocytopenia” or “lymphocytic leucopenia”interchangeably refer to an abnormally small number of lymphocytes inthe circulating blood or in peripheral circulation. Quantitatively,lymphopenia can be described by various cutoffs. In some embodiments, apatient is suffering from lymphopenia when their circulating blood totallymphocyte count falls below about 600/mm³. In some embodiments, apatient suffering from lymphopenia has less than about 2000/μL totalcirculating lymphocytes at birth, less than about 4500/μL totalcirculating lymphocytes at about age 9 months, or less than about1000/μL total circulating lymphocytes patients older than about 9 months(children and adults). Lymphocytopenia has a wide range of possiblecauses, including viral (e.g., HIV infection), bacterial (e.g., activetuberculosis infection), and fungal infections; chronic failure of theright ventricle of the heart, Hodgkin's disease and cancers of thelymphatic system, leukemia, a leak or rupture in the thoracic duct, sideeffects of prescription medications including anticancer agents,antiviral agents, and glucocorticoids, malnutrition resulting from dietsthat are low in protein, radiation therapy, uremia, autoimmunedisorders, immune deficiency syndromes, high stress levels, and trauma.Lymphopenia may also be of unknown etiology (i.e., idiopathiclymphopenia). Peripheral circulation of all types of lymphocytes orsubpopulations of lymphocytes (e.g., CD4+ T cells) may be depleted orabnormally low in a patient suffering from lymphopenia. See, e.g., TheMerck Manual, 18^(th) Edition, 2006, Merck & Co.

The term “native mammalian interleukin-15 (IL-15)” refers to anynaturally occurring interleukin-15 nucleic acid and amino acid sequencesof the IL-15 from a mammalian species. Those of skill in the art willappreciate that interleukin-15 nucleic acid and amino acid sequences arepublicly available in gene databases, for example, GenBank through theNational Center for Biotechnological Information on the worldwide web atncbi.nlm.nih.gov. Exemplified native mammalian IL-15 nucleic acid oramino acid sequences can be from, for example, human, primate, canine,feline, porcine, equine, bovine, ovine, rodentia, murine, rat, hamster,guinea pig, etc. Accession numbers for exemplified native mammalianIL-15 nucleic acid sequences include NM_172174.2 (human preproprotein);NM_172175 (human); NM_000585.3 (human preproprotein); U19843 (macaque);DQ021912 (macaque); AB000555 (macaque); NM_214390 (porcine); DQ152967(ovine); NM_174090 (bovine); NM_008357 (murine); NM_013129 (rattus);DQ083522 (water buffalo); XM_844053 (canine); DQ157452 (lagomorpha); andNM_001009207 (feline). Accession numbers for exemplified nativemammalian IL-15 amino acid sequences include NP_000576.1 (humanpreproprotein); NP_751914 (human preproprotein); CAG46804 (human);CAG46777 (human); AAB60398 (macaque); AAY45895 (macaque); NP_999555(porcine); NP_776515 (bovine); AAY83832 (water buffalo); ABB02300(ovine); XP_849146 (canine); NP_001009207 (feline); NP_037261 (rattus);and NP_032383 (murine).

The term “interleukin-15” or “IL-15” refers to a polypeptide that has atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to a native mammalian IL-15 amino acid sequence, or anucleotide encoding such a polypeptide, is biologically active, meaningthe mutated protein (“mutein”) has functionality similar (75% orgreater) to that of a native IL-15 protein in at least one functionalassay. Functionally, IL-15 is a cytokine that regulates T cell andnatural killer cell activation and proliferation. IL-15 and IL-2 sharemany biological activities, including binding to CD122, the IL-2β/IL-15βreceptor subunit. The number of CD8+ memory cells is controlled by abalance between this IL-15 and IL-2. IL-15 induces the activation of JAKkinases, as well as the phosphorylation and activation of transcriptionactivators STAT3, STAT5, and STAT6. IL-15 also increases the expressionof apoptosis inhibitor BCL2L1/BCL-x(L), possibly through thetranscription activation activity of STAT6, and thus prevents apoptosis.Two alternatively spliced transcript variants of the IL-15 gene encodingthe same mature protein have been reported. Exemplified functionalassays of an IL-15 polypeptide include proliferation of T-cells (see,for example, Montes, et al., Clin Exp Immunol (2005) 142:292), andactivation of NK cells, macrophages and neutrophils. Methods forisolation of particular immune cell subpopulations and detection ofproliferation (i.e., ³H-thymidine incorporation) are well known in theart. Cell-mediated cellular cytotoxicity assays can be used to measureNK cell, macrophage and neutrophil activation. Cell-mediated cellularcytotoxicity assays, including release of isotopes (⁵¹Cr), dyes (e.g.,tetrazolium, neutral red) or enzymes, are also well known in the art,with commercially available kits (Oxford Biomedical Research, Oxford, M;Cambrex, Walkersville, Md.; Invitrogen, Carlsbad, Calif.). IL-15 hasalso been shown to inhibit Fas mediated apoptosis (see, Demirci and Li,Cell Mol Immunol (2004) 1:123). Apoptosis assays, including for example,TUNEL assays and annexin V assays, are well known in the art withcommercially available kits (R&D Systems, Minneapolis, Minn.). See also,Coligan, et al., Current Methods in Immunology, 1991-2006, John Wiley &Sons.

The term “native mammalian interleukin-15 Receptor alpha (IL15Rα)”refers to any naturally occurring interleukin-15 receptor alpha nucleicacid and amino acid sequences of the IL-15 receptor alpha from amammalian species. Those of skill in the art will appreciate thatinterleukin-15 receptor alpha nucleic acid and amino acid sequences arepublicly available in gene databases, for example, GenBank through theNational Center for Biotechnological Information on the worldwide web atncbi.nlm.nih.gov. Exemplified native mammalian IL-15 receptor alphanucleic acid or amino acid sequences can be from, for example, human,primate, canine, feline, porcine, equine, bovine, ovine, rodentia,murine, rat, hamster, guinea pig, etc. Accession numbers for exemplifiednative mammalian IL-15 nucleic acid sequences include NM_172200.1 (humanisoform 2); and NM_002189.2 (human isoform 1 precursor). Accessionnumbers for exemplified native mammalian IL-15 amino acid sequencesinclude NP_751950.1 (human isoform 2); and NP_002180.1 (human isoform 1precursor).

The term “interleukin-15 receptor alpha” or “IL15Rα” refers to apolypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to a native mammalian IL15Rα amino acidsequence, or a nucleotide encoding such a polypeptide, is biologicallyactive, meaning the mutated protein (“mutein”) has functionality similar(75% or greater) to that of a native IL15Rα protein in at least onefunctional assay. IL15Rα is a cytokine receptor that specifically bindsIL15 with high affinity. One functional assay is specific binding to anative IL-15 protein.

The term “soluble IL-15 Receptor alpha” or “sIL-15α” refers to forms ofIL-15 Receptor alpha lacking the transmembrane anchor portion of thereceptor and thus able to be secreted out of the cell without beinganchored to the plasma membrane. Exemplary sIL-15α include aa 31-205 andaa31-185 of the native IL-15 Receptor alpha.

An “IL-15Rα Fc fusion” or an “IL-15Rα fused to an Fc region” as usedherein refers to forms of IL-15Rα in which the protein is fused to oneor more domains of an Fc region of an immunoglobulin, typically of anIgG immunoglobulin. The Fc region comprises the CH2 and CH3 domains ofthe IgG heavy chain and the hinge region. The hinge serves as a flexiblespacer between the two parts of the Fc-Fusion protein, allowing eachpart of the molecule to function independently. The use of Fc fusions isknown in the art (see, e.g., U.S. Pat. Nos. 7,754,855; 5,480,981;5,808,029; WO7/23614; WO98/28427 and references cited therein. Fc fusionproteins can include variant Fc molecules (e.g., as described in U.S.Pat. No. 7,732,570). Fc fusion proteins can be soluble in the plasma orcan associate to the cell surface of cells having specific Fc receptors.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. The term encompasses nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,which have similar binding properties as the reference nucleic acid, andwhich are metabolized in a manner similar to the reference nucleotides.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Degenerate codon substitutions can beachieved by generating sequences in which the third position of one ormore selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is usedinterchangeably with gene, cDNA, mRNA, oligonucleotide, andpolynucleotide.

Degenerate codon substitutions for naturally occurring amino acids arein Table 1.

TABLE 1 1^(st) position 2^(nd) position 3^(rd) position (5′ end) U(T) CA G (3′ end) U(T) Phe Ser Tyr Cys U(T) Phe Ser Tyr Cys C Leu Ser STOPSTOP A Leu Ser STOP Trp G C Leu Pro His Arg U(T) Leu Pro His Arg C LeuPro Gln Arg A Leu Pro Gln Arg G A Ile Thr Asn Ser U(T) Ile Thr Asn Ser CIle Thr Lys Arg A Met Thr Lys Arg G G Val Ala Asp Gly U(T) Val Ala AspGly C Val Ala Glu Gly A Val Ala Glu Gly G

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region(e.g., of a IL-15 or IL-15Rα sequence), when compared and aligned formaximum correspondence over a comparison window or designated region) asmeasured using a BLAST or BLAST 2.0 sequence comparison algorithms withdefault parameters described below, or by manual alignment and visualinspection (see, e.g., NCBI web site or the like). Such sequences arethen said to be “substantially identical.” This definition also refersto, or can be applied to, the compliment of a test sequence. Thedefinition also includes sequences that have deletions and/or additions,as well as those that have substitutions. As described below, thepreferred algorithms can account for gaps and the like. Preferably,identity exists over a region that is at least about 25, 50, 75, 100,150, 200 amino acids or nucleotides in length, and oftentimes over aregion that is 225, 250, 300, 350, 400, 450, 500 amino acids ornucleotides in length or over the full-length of am amino acid ornucleic acid sequences.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared (here, an entire “nativemammalian” IL-15 amino acid or nucleic acid sequence). When using asequence comparison algorithm, test and reference sequences are enteredinto a computer, subsequence coordinates are designated, if necessary,and sequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A preferred example of algorithm that is suitable for determiningpercent sequence identity and sequence similarity are the BLASTalgorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410(1990), respectively. BLAST software is publicly available through theNational Center for Biotechnology Information on the worldwide web atncbi.nlm.nih.gov/. Both default parameters or other non-defaultparameters can be used. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc.Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

The term “GC content” refers to the percentage of a nucleic acidsequence comprised of deoxyguanosine (G) and/or deoxycytidine (C)deoxyribonucleosides, or guanosine (G) and/or cytidine (C)ribonucleoside residues.

The term “operably linked” refers to a functional linkage between afirst nucleic acid sequence and a second nucleic acid sequence, suchthat the first and second nucleic acid sequences are transcribed into asingle nucleic acid sequence. Operably linked nucleic acid sequencesneed not be physically adjacent to each other. The term “operablylinked” also refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a transcribable nucleic acidsequence, wherein the expression control sequence directs transcriptionof the nucleic acid corresponding to the transcribable sequence.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” as used herein applies to amino acidsequences. One of skill will recognize that individual substitutions,deletions or additions to a nucleic acid, peptide, polypeptide, orprotein sequence which alters, adds or deletes a single amino acid or asmall percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   -   1) Alanine (A), Glycine (G);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);    -   7) Serine (S), Threonine (T); and    -   8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins        (1984)).

The terms “mammal” or “mammalian” refer to any animal within thetaxonomic classification mammalia. A mammal can refer to a human or anon-human primate. A mammal can refer to a domestic animal, includingfor example, canine, feline, rodentia, including lagomorpha, murine,rattus, Cricetinae (hamsters), etc. A mammal can refer to anagricultural animal, including for example, bovine, ovine, porcine,equine, etc.

The term “therapeutically effective amount” refers to the dose of atherapeutic agent or agents sufficient to achieve the intendedtherapeutic effect with minimal or no undesirable side effects. Atherapeutically effective amount can be readily determined by a skilledphysician, e.g., by first administering a low dose of thepharmacological agent(s) and then incrementally increasing the doseuntil the desired therapeutic effect is achieved with minimal or noundesirable side effects.

The term “supraphysiologic levels” refers to levels of IL-15 in aparticular tissue, e.g., blood, plasma, serum, thymus, that are abovenaturally occurring physiologic levels. Supraphysiologic levels of IL-15in a tissue can also be achieved when the concentration of IL-15 in thattissue is sustained above naturally occurring levels for an extendedperiod of time, e.g., for consecutive days or weeks or for the durationof therapeutic treatment. For example, IL-15 DNA or protein can beadministered at a dose sufficient to achieve plasma levels of IL-15 ofabout 1 to 1000 ng/ml, for example, plasma levels of IL-15 of about 10to 1000 ng/ml. The IL-15 and IL-15Rα can be delivered in equimolaramounts. Alternatively, an IL-15/IL-15Rα protein complex can beadministered at a dose of about 0.01 to 0.5 mg/kg.

The term “co-administer” refers to the presence of two pharmacologicalagents, e.g., IL-15 and IL-15Rα, in the blood at the same time. The twopharmacological agents can be administered concurrently or sequentially.

The term “consisting essentially of” refers to administration of thepharmacologically active agents expressly recited, e.g., IL-15 andIL-15Rα, and excludes pharmacologically active agents not expresslyrecited, e.g., an antigen. The term consisting essentially of does notexclude pharmacologically inactive or inert agents, e.g.,physiologically acceptable carriers or excipients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic of the mutual stabilization of IL-15 andIL-15α.

FIG. 2 illustrates the effects of systemic co-administration ofpolynucleotides expressing IL-15 and IL-15Rα on spleen weight (toppanel), thymus weight (middle panel) and percentage of lymphocytes inthe bone marrow (bottom panel).

FIG. 3 illustrates the effects of systemic co-administration ofpolynucleotides expressing IL-15 and IL-15Rα on T cell maturation in thethymus. Double positive CD4+CD8+ T cells are decreased with aconcomitant increase in CD3high single positive T cells (i.e., CD4+ orCD8+ T cells).

FIG. 4 illustrates the migration of dividing carboxyfluoresceinsuccinimidyl ester (“CFSE”)-loaded thymocytes to the lung inIL-15-treated and untreated control mice (upper panels). The lowerpanels show increased expression of CD122 (IL-2Rβ/IL-15Rβ) onlymphocytes, e.g., total T cells and CD+ T cells, in the lung.

FIG. 5 illustrates lymphocyte reconstitution in lung tissue of IL-15knockout (KO) mice treated with plasmid DNA encoding IL-15/IL-15Rαcompared to untreated control KO mice.

FIG. 6 provides a schematic of the time course of a lymphodepletionexperiment.

FIG. 7 illustrates spleen weight over time after cyclophosphamide (Cyp)and Cyp+IL-15/IL-15Rα administration.

FIG. 8 illustrates the increase in lung NK cells after Cypadministration.

FIG. 9 illustrates the increase in lung T cells in the presence ofIL-15/IL-15Rα.

FIG. 10 illustrates that CD8+ T cells partially recover afterIL-15/IL-15Rα administration.

FIG. 11 illustrates the increase in lung CD8+ T cells in the presence ofIL-15/IL-15Rα as reflected in the change of the ratio of CD8+ to CD4+ Tcells after IL-15 administration.

FIG. 12 illustrates a T cell analysis in the spleen after Cyp andIL-15/IL-15Rα administration.

FIG. 13 illustrates the full recovery of bone marrow T cells afterIL-15/IL-15Rα administration.

FIG. 14 illustrates the IL-15/IL-15Rα treatment protocol for lymphopenicmice used in Example 3.

FIG. 15 illustrates that a single administration ofIL-15/IL-15sRα-encoding DNA is sufficient for the complete recovery ofNK cells in spleen and lung 5 days after DNA injection.

FIG. 16 illustrates that IL-15/IL-15sRα administration promotes therecovery of CD8 T cells within 10 days after treatment, withoutsignificantly affecting the recovery of CD4 T cells.

FIG. 17 illustrates that high levels of circulating IL-15/IL-15sRαpromote a transient increase in the Teffector/Treg ratio afterlymphoablation.

FIG. 18 illustrates IL-15 levels in serum following hydrodynamicdelivery of DNA vectors expressing different forms of IL-15.

FIG. 19 illustrates CD25 expression on the surface of spleen T cellsafter IL-15/IL-15Rα DNA delivery.

FIG. 20 illustrates expression of CD62L on the surface of spleen T cellsafter IL-15/IL-15Rα DNA delivery.

FIG. 21 illustrates express of CD44 on the surface of spleen T cellsafter IL-15/IL-5Rα DNA delivery.

FIG. 22 illustrates a protocol (Example 5) for administration ofpurified IL-15/IL-15sRα in vivo.

FIG. 23 illustrates that purified IL-15/IL-15Rα is bioactive in vivo.

DETAILED DESCRIPTION

1. Introduction

The present invention is based, in part, on the surprising discoverythat subjecting thymic tissue to supraphysiological levels of IL-15promotes the maturation of T cells in the thymus from double positiveCD4+CD8+ T cells to single positive (i.e., CD4+ or CD8+) CD3high Tcells, decreases the frequency of apoptotic thymocytes, and increasesthe migration of mature T cells from the thymus to peripheral tissues,including lymphoid and non-lymphoid peripheral tissues.

The present invention is further based, in part, on the surprisingdiscovery that systemic administration of supraphysiological levels ofIL-15 promotes the maturation and export of lymphocytes from centrallymphoid tissues (e.g., in the thymus and bone marrow) to peripheraltissues, including lymphoid and non-lymphoid peripheral tissues.

2. Methods of Promoting Maturation of Lymphocytes in a Central LymphoidOrgan and the Migration of the Lymphocytes to Peripheral Tissues

The present invention provides methods of promoting T cell maturation inthe thymus, decreasing apoptosis of T cells in the thymus and promotingmigration or output of mature T cells from the thymus, by contacting thethymus tissue with supraphysiological levels of IL-15. The thymic tissuecan be in vivo or in vitro.

When the IL-15 is administered in vivo, it is provided to a subject orpatient or individual in need thereof. The subject can be any mammal. Insome embodiments, the mammal is a human or a non-human primate. Subjectswho will benefit from the present methods have a deficiency of maturethymocytes and/or other lymphocytes in peripheral tissues, includinglymphoid and non-lymphoid peripheral tissues. In some embodiments, thesubject is immunodeficient or has lymphopenia. In some embodiments, thesubject has a drug-induced immunodeficiency, e.g., due to anticancerdrugs. In some embodiments, the subject has an immunodeficiencysecondary to a disease, e.g., HIV infection. In some embodiments, thesubject may have a genetic mutation that results in a non-functionalIL-15 or non-functional IL-15 receptor subunit (e.g., IL-15Rα, IL-15Rβ,or IL-15Rγ).

Sustained exposure of thymic tissue to supraphysiological levels ofIL-15 promotes the maturation of double positive T cells. IL-15 promotesthe terminal differentiation of the thymocytes to single positive Tcells expressing either CD4 or CD8. The mature T cells also may expressCD122 (also known as the beta subunit of IL-2/IL-15 receptor). Themature T cells may also express high levels of the CD3 surface protein.IL-15-induced maturation of T cells also corresponds to a reduction inthe frequency of immature T cells that undergo apoptosis. By contactingthe thymic tissue with supraphysiologic levels of IL-15, the CD4+CD8+double positive and CD3low T cells can be substantially eliminated asthe cells mature into single positive CD3high T cells. After exposure tosupraphysiologic levels of IL-15, at least 60%, 70%, 80%, 90%, 95% ormore of the T cells are CD4+ or CD8+ single positive CD3high T cells.

IL-15-induced maturation of T cells in thymus tissue also promotes themigration of the mature T cells to the peripheral tissues, includinglymphoid and non-lymphoid peripheral tissues. The mature T cells leavingthe thymus may or may not be activated. For example, after about 2, 3,4, 5, 6, 7, 8, 9, 10 or more days exposure to supraphysiologic levels ofIL-15, the thymus organ may have decreased in size, e.g., by at leastabout 30%, 40%, 50%, or more, due to IL-15-induced thymic output.

Systemic administration of supraphysiologic levels of IL-15, e.g.,sustained over the course of e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore days, also promotes the maturation and migration of lymphocytes,including NK cells, from bone marrow. For example, after about 2, 3, 4,5, 6, 7, 8, 9, 10 or more days exposure to supraphysiologic levels ofIL-15, the percentage of lymphocytes in the bone marrow may havedecreased, e.g., by at least about 50%, 60%, 70%, 80% or more, due toIL-15-induced lymphocyte output from bone marrow.

At the same time that the number of lymphocytes decrease in the centrallymphoid tissues, i.e., in the thymus and bone marrow, the number oflymphocytes in peripheral lymphoid tissues, e.g., spleen, lymph node,mucosal-associated lymphoid tissues (MALT), e.g., tonsils and/orgut-associated lymphoid tissues (GALT), including Peyer's patches,increases. Furthermore, the number of lymphocytes in peripheralnon-lymphoid tissues, including the lung, liver, kidney, skin, and othertissues, also increases. In some embodiments, the administration ofsupraphysiologic levels of IL-15 increases the number of lymphocytes,including T cells, B cells and NK cells, in the blood.

3. Methods of Treating Lymphopenia

As explained above, in one aspect, the invention is based on thediscovery that systemic administration of supraphysiological levels ofIL-15 promotes the maturation and export of lymphocytes from centrallymphoid tissues (e.g., in the thymus and bone marrow) to peripheraltissues, including lymphoid and non-lymphoid peripheral tissues.

Accordingly, the invention provides methods for preventing, reducing andinhibiting the depletion of lymphocytes, including T cells, B cells andnatural killer (NK) cells, in peripheral circulation or tissues bysystemic administration of IL-15 to a subject in need thereof. Thepresent invention also provides methods for accelerating the recoveryfrom and shortening the time period of depletion of lymphocytes,including T cells, B cells and natural killer (NK) cells, in peripheralcirculation or tissues by systemic administration of IL-15 to a subjectin need thereof.

The subject, patient or individual can be any mammal. In someembodiments, the mammal is a human or a non-human primate. In someembodiments, the individual is a domestic mammal (e.g., a canine orfeline), a laboratory mammal (e.g., a mouse, a rat, a rabbit, ahamster), or an agricultural mammal (e.g., a bovine, a porcine, a ovine,an equine). Subjects who will benefit from the present methods eitheralready have or will have (e.g., as a result of a course of drugtreatment) a deficiency of mature lymphocytes in peripheral circulationor tissues, including lymphoid and non-lymphoid peripheral tissues. Insome embodiments, the subject is immunodeficient or has lymphopenia. Forthe purposes of treatment, the patient is already suffering abnormallylow levels of circulating lymphocytes. For the purposes of prevention,the patient may have normal levels of peripheral lymphocytes and islikely to experience lymphodepletion, e.g., as a result of achemotherapeutic treatment.

Standards for diagnosing lymphopenia are known in the art, and can bemade by any trained physician. In some embodiments, the patient has acirculating blood total lymphocyte count that is below about 600/mm³. Insome embodiments, the patient has a circulating blood total lymphocytecount that is less than about 2000/μL total circulating lymphocytes atbirth, less than about 4500/μL total circulating lymphocytes at aboutage 9 months, or less than about 1000/μL total circulating lymphocytespatients older than about 9 months (children and adults). See, e.g., TheMerck Manual, 18th Edition, 2006, Merck & Co.

The origins or etiology of the depletion or abnormally low can be forany reason. Lymphocytopenia has a wide range of possible causes,including viral (e.g., HIV infection), bacterial (e.g., activetuberculosis infection), and fungal infections; chronic failure of theright ventricle of the heart, Hodgkin's disease and cancers of thelymphatic system, leukemia, a leak or rupture in the thoracic duct, sideeffects of prescription medications including anticancer agents,antiviral agents, and glucocorticoids, malnutrition resulting from dietsthat are low in protein, radiation therapy, uremia, autoimmunedisorders, immune deficiency syndromes, high stress levels, and trauma.The lymphopenia may also be of unknown etiology (i.e., idiopathiclymphopenia).

The lymphocyte depletion may involve total lymphocytes (e.g., T cells, Bcells, and NK cells, etc.), or may only involve a subpopulation of totallymphocytes (one or more of T cells, CD4+ T cells, CD8+ T cells, Bcells, NK cells).

In some embodiments, the patient has a disease that causes depletion ofperipheral circulating lymphocytes. For example, the patient may sufferfrom a cancer, including Hodgkin's disease and cancers of the lymphaticsystem, leukemia; a viral infection, including HIV or hepatitis virus.In some embodiments, the patient is receiving chemotherapy, e.g., ananticancer agent, an antiviral or antiretroviral agent, or aglucocorticoid, that causes depletion of peripheral circulatinglymphocytes. Exemplary pharmacological agents that can causelymphodepletion include without limitation vinblastine, fludarabine,aclarubicin, doxorubicin, exemestane, alefacept, alemtuzumab,chloramphenicol, pamidronate, idarubicin and cyclophosphamide.

In some embodiments, the subject may have a genetic mutation thatresults in a non-functional IL-15 or non-functional IL-15 receptorsubunit (e.g., IL 15Rα, IL 15Rβ, or IL 15Rγ).

4. IL-15

The IL-15 for use in the invention can be any physiologically active(i.e., functional) IL-15. The IL-15 can be delivered as a polypeptide ora polynucleotide encoding IL-15. The IL-15 can be full-length or aphysiologically active fragment thereof, for example, an IL-15 fragmentthat retains binding to IL-15Rα and/or IL-15Rβ, or an IL-15 fragmentthat promotes proliferation and/or maturation of T cells. In someembodiments, the delivered or expressed IL-15 polypeptide has one ormore amino acids that are substituted, added or deleted, while stillretaining the physiological activity of IL-15. In some embodiments, thedelivered or expressed IL-15 shares at least 90%, 93%, 95%, 97%, 98%,99% or 100% amino acid sequence identity with a wild-type IL-15, e.g.,SEQ ID NO:2. In some embodiments, the polynucleotide encoding IL-15shares at least 90%, 93%, 95%, 97%, 98%, 99% or 100% nucleic acidsequence identity with a wild-type IL-15 coding sequence, e.g., SEQ IDNO:1.

The polynucleotide encoding IL-15 may have one or more codons alteredfor improved expression. In some embodiments, the polynucleotideencoding IL-15 shares at least 90%, 93%, 95%, 97%, 98%, 99% or 100%nucleic acid sequence identity with a wild-type IL-15 coding sequence,e.g., SEQ ID NO:3. In some embodiments, the polynucleotide encodingIL-15 shares at least 96%, 97%, 98%, 99% or 100% nucleic acid sequenceidentity with a wild-type IL-15 coding sequence, e.g., SEQ ID NO:4.Polynucleotides encoding IL-15 which have altered codons for improvedexpression are described, e.g., in WO 2007/084342 and in WO 2004/059556,the entire disclosures of each of which are hereby incorporated hereinby reference for all purposes.

The polynucleotide encoding IL-15 can be operably linked topolynucleotide encoding a native signal peptide sequence, e.g., the longIL-15 signal peptide sequence (LSP) or the short IL-15 signal peptidesequence (SSP). In some embodiments, the nucleic acid sequence encodinga native IL-15 signal peptide is replaced with a nucleic acid sequenceencoding a signal peptide from a heterologous protein. The heterologousprotein can be, for example, from tissue plasminogen activator (tPA),growth hormone, granulocyte-macrophage colony stimulating factor(GM-CSF) or an immunoglobulin (e.g., IgE). An example of a humanGMCSF-IL-15 fusion is provided in SEQ ID NO:18. In some embodiments, thenucleic acid encoding the IL-15 is operably linked to a nucleic acidencoding an RNA export element, for example a CTE or RTEm26CTE.

Preferably, the IL-15 is administered as a heterodimer with IL-15Rα. Oneor both of the IL-15 and the IL-15Rα can be delivered as a polypeptide.One or both of the IL-15 and the IL-15Rα can be delivered as apolynucleotide. In one embodiment, the IL-15 and the IL-15Rα areco-administered as polypeptides. In one embodiment, an IL-15 polypeptideis co-administered with a polynucleotide encoding IL-15Rα. In oneembodiment, an IL-15Rα polypeptide is co-administered with apolynucleotide encoding IL-15.

The administered IL-15Rα can be any physiologically active (i.e.,functional) IL-15Rα. The IL-15Rα can be delivered as a polypeptide or apolynucleotide encoding IL-15Rα. The IL-15Rα can be full-length or aphysiologically active fragment thereof, for example, an IL-15Rαfragment that retains specific binding to IL-15. Further, the IL-15Rα,e.g., a fragment that retains specific binding to IL-15 and lacks thetransmembrane anchor region, can be fused to an Fc region. In someembodiments, the delivered or expressed IL-15Rα polypeptide has one ormore amino acids that are substituted, added or deleted, while stillretaining the physiological activity of IL-15Rα. In some embodiments,the delivered or expressed IL-15 shares at least 90%, 93%, 95%, 97%,98%, 99% or 100% amino acid sequence identity with a wild-type IL-15Rα,e.g., SEQ ID NO:5 or SEQ ID NO:7. In some embodiments, thepolynucleotide encoding IL-15 shares at least 90%, 93%, 95%, 97%, 98%,99% or 100% nucleic acid sequence identity with a wild-type IL-15 codingsequence, e.g., SEQ ID NO:6 or SEQ ID NO:8.

The polynucleotide encoding IL-15Rα may have one or more codons alteredfor improved expression. In some embodiments, the polynucleotideencoding IL-15Rα shares at least 90%, 93%, 95%, 97%, 98%, 99% or 100%nucleic acid sequence identity with a wild-type IL-15Rα coding sequence,e.g., SEQ ID NO:9 or SEQ ID NO:11. Polynucleotides encoding IL-15Rαwhich have altered codons for improved expression are described, e.g.,in WO 2007/084342.

The polynucleotide encoding IL-15Rα can be operably linked topolynucleotide encoding a native signal peptide sequence. In someembodiments, the nucleic acid sequence encoding a native IL-15Rα signalpeptide is replaced with a nucleic acid sequence encoding a signalpeptide from a heterologous protein. The heterologous protein can be,for example, from tissue plasminogen activator (tPA), growth hormone,granulocyte-macrophage colony stimulating factor (GM-CSF) or animmunoglobulin (e.g., IgE). In some embodiments, the nucleic acidencoding the IL-15Rα is operably linked to a nucleic acid encoding anRNA export element, for example a CTE or RTEm26CTE.

In some embodiments, the IL-15Rα can be in the form of an Fc fusionprotein. Examples of sIL-15Rα polypeptide sequences are shown in SEQ IDNO:17 and SEQ ID NO:20. Typically, such proteins are secreted and can befound soluble in the plasma, or they can be associated with the surfaceof cells expressing the Fc receptor for the Fc region of the fusionprotein. Different fragments of IL-15Rα can be fused to the Fc region.Two examples of functional fusions are provided as SEQ ID NO:17 and SEQID NO:20, containing 205 or 200 amino acids within the IL-15Rα region.In some embodiments, the IL-15Rα region of the fusion protein can bereleased by proteolytic cleavage. In some embodiments, I-L15Rαfunctional region of the protein is linked to a polypeptide that is ableto bind specific cell types via surface receptors. In some embodiments,the IL15-Rα Fc fusion protein shares at least 95%, 96%, 97%, 98%, 99% or100% amino acid sequence identity with a polypeptide selected from thegroup consisting of SEQ ID NO:17 and SEQ ID NO:20.

In some embodiments, a polynucleotide encoding IL-15 is co-administeredwith a polynucleotide encoding IL-15Rα. The polynucleotide encodingIL-15 and the polynucleotide encoding IL-15Rα can be administered on thesame vector or on separate vectors. Preferably the polynucleotideencoding IL-15 is co-administered with a polynucleotide encoding IL-15Rαare on the same vector. An example of a plasmid that encodes anIL-15Rα-Fc fusion having a polypeptide sequence of SEQ ID NO:17 and ahuman GM-CSF signal peptide-IL-15 of SEQ ID NO:18 is provided in SEQ IDNO:16. A second example of a plasmid that encodes an IL-15Rα-Fc fusionhaving a polypeptide sequence of SEQ ID NO:20 and a human GM-CSF signalpeptide-IL-15 of SEQ ID NO:18 is provided in SEQ ID NO:19. In someembodiments, the administered vector shares at least 95%, 97%, 98%, 99%or 100% nucleic acid sequence identity with a plasmid vector selectedfrom the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, and SEQ ID NO:19.

It is understood by one skilled in the art that expression vectors,promoters, polyadenylation signals, and secretory peptides alternativesto those in the example sequences provided herein can be used for theexpression of the optimized IL-15 and IL-15 Receptor alpha.

For the purposes of the present methods, the IL-15 is not being used asan adjuvant to enhance the immune response against a particular antigen.Therefore, in the present methods, the IL-15 is administered without anantigen. Stated another way, the IL-15 is not co-administered with anantigen.

The IL-15 (and the IL-15Rα) are administered at a dose sufficient toachieve supraphysiological levels of IL-15 systemically or in the targettissue, e.g., thymus, for the desired time period. The desired timeperiod can be hours, days, weeks, or longer if necessary. In someembodiments, supraphysiological levels of IL-15 are sustained throughoutthe duration of treatment or until a desired therapeutic endpoint isachieved, e.g., the repopulation of peripheral tissues with lymphocytes.In some embodiments, the IL-15 is administered one time, as a bolus. Insome embodiments, the IL-15 is administered two or more times. Whenadministered multiple times, the IL-15 can be administered daily,weekly, bi-weekly, monthly, or as needed to sustain supraphysiologicallevels of IL-15 systemically or in the target tissue.

In embodiments where the IL-15 (and the IL-15Rα) are administered as apolypeptide, typical dosages can range from about 0.1 mg/kg body weightup to and including about 0.5 mg/kg body weight. In some embodiments,the dose of polypeptide is about 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.3,0.4, 0.5 mg/kg body weight.

In embodiments where the IL-15 (and the IL-15Rα) are administered as apolynucleotide, dosages are sufficient to achieve plasma levels of IL-15of about 1 to 1000 ng/ml, for example, plasma levels of IL-15 of about10 to 1000 ng/ml. Such a range of plasma concentrations can be achieved,e.g., after intramuscular electroporation of about 0.1 mg IL-15/IL-15sRαexpressing DNA plasmid per kg body weight. In some embodiments, the doseof nucleic acid is about 0.02, 0.05, 0.1, 0.2, 0.5 mg/kg body weight.

The IL-15 can be administered by a route appropriate to effect systemicsupraphysiological levels of IL-15 or supraphysiological levels of IL-15in the target tissue, e.g., thymus. When co-administered with IL-15Rα,the IL-15 and the IL-15Rα can be administered via the same or differentroutes. In some embodiments, the IL-15 (and the IL-15Rα) areadministered systemically, including without limitation, enterally(i.e., orally) or parenterally, e.g., intravenously, intramuscularly,subcutaneously, intradermally, intranasally, or inhalationally. In someembodiments, the IL-15 (and the IL-15Rα) are administered locally, forexample, intrathymically or directly into the bone marrow.

For treatment of lymphopenia, systemic administration of IL-15 promotesand accelerates the repopulation of peripheral lymphocyte populations.After administration of IL-15, the peripherally circulating lymphocytesor lymphocyte subpopulations can be at least 80%, 85%, 90% or 95% oflevels considered to be normal in a healthy individual. In someembodiments, the lymphocytes or lymphocyte subpopulations are completelyrepopulated to normal levels. In some embodiments, the repopulation oflymphocytes is days or weeks faster in an individual who receivedadministration of IL-15 in comparison to an individual who did notreceive administration of IL-15.

Systemic administration of IL-15 also prevents, reduces or inhibitslymphocyte depletion in peripheral circulation, e.g., caused bychemotherapy or radiation therapy. After administration of IL-15, theperipherally circulating lymphocytes or lymphocyte subpopulations can bemaintained at levels of at least 70%, 75%, 80%, 85%, 90% or 95% ofnormal levels. In some embodiments, the lymphocytes or lymphocytesubpopulations are maintained at normal levels.

In some embodiments, the IL-15 is co-administered with achemotherapeutic agent that causes or may cause lymphopenia orlymphocyte depletion in peripheral tissues. The chemotherapeutic agentmay be an anticancer agent or an antiviral agent. In some embodiments,the IL-15 is administered after a course of treatment with achemotherapeutic agent that causes or may cause lymphopenia orlymphocyte depletion in peripheral tissues. In some embodiments, theIL-15 is administered prior to, during or after a course of radiationtherapy.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Systemic Administration of IL-15 Promotes Maturation of TCells in the Thymus and the Migration of T Cells to Peripheral Tissues

IL-15/IL-15Rα DNA was expressed systemically and locally at variouslevels in either normal or IL-15 knockout (KO) mice to furtherunderstand IL-15 biology. See, Bergamaschi, et al., (2008) J Biol Chem283:4189-4199. Supraphysiologic levels of IL-15/IL-15Rα in normal micehave rapid and profound effects in many tissues. There is a rapid andreversible increase in the size of spleen, whereas the thymus becomessmaller and bone marrow lymphocyte numbers decrease (FIG. 2). We havepreviously shown that spleen and lymph node size increase isproportional to the amount of IL-15 in the plasma. See, Bergamaschi, etal., (2008) J Biol Chem 283:4189-4199. The kinetics and composition oflymphocytes in many tissues were studied using 10 parameter flowcytometry, as well as adoptive transfer of cells and in vivo labeling.Our results underscore the strong effects of IL-15 at all steps oflymphocyte development, as also suggested by many investigators.Reviewed in, e.g., Boyman, et al., (2007) Curr Opin Immunol 19:320-326:Sprent, et al., (2008) Immunol Cell Biol 86:312-319; Sprent and Surh,(2003) Immunol Lett 85:145-149; Surh, et al., (2006) Immunol Rev211:154-163; Surh and Sprent, (2005) Semin Immunol 17:183-191; and Surhand Sprent, (2008) Immunity 29:848-862. However, prior to the presentinvention, the effects of IL-15 in the thymus have not been elucidated.Our results indicate that IL-15 stimulates the maturation of CD4+CD8+double positive thymocytes into CD3high single positive T cells (FIG. 3)and accelerates their rapid migration to the periphery (FIG. 4). Sevendays after in situ labeling of thymocytes, IL-15/IL-15Rα promoted theirmigration to the lung. In the presence of IL-15/IL-15Rα the lymphocytesin the lung have higher levels of IL-2/IL-15Rα (CD122, see, FIG. 4,bottom) indicating that they are activated. These results are consistentwith the notion that IL-15 promotes not only accelerated exit from thethymus, but also the migration to peripheral tissues and the activationof these lymphocytes.

Our results also show that, in addition to NK and memory CD8+ T cellsthat are profoundly affected, as expected, all lymphocytes includingnaïve and memory CD4 and CD8 cells, and B lymphocytes are also affectedto either divide, migrate or be activated. This is in agreement with thewidespread (but not universal) expression of the IL-2/IL-15 betagammareceptor. The hierarchy of responsiveness of the lymphocyte subsets toIL-15 reflects the levels of CD122 (IL-2Rbeta) on their surface. See,Bergamaschi, et al., (2008) J Biol Chem 283:4189-4199.

Our observations are further supported by experiments performed in anIL-15 KO model, to correct the lymphocyte defects by administeringplasmid DNA encoding IL-15/IL-15Rα heterodimer. IL-15 KO mice arecharacterized by a decrease in total T cell count that preferentiallyaffects CD8+ T cells, which are almost completely absent in peripheraltissues. We show that IL-15/IL-15Rα is able to repopulate non-lymphoidorgans, such as lungs, with both mature CD4 and CD8 T lymphocytes. Theincrease in CD4 T cells upon IL-15/IL-15Rα treatment is 10-fold, whilethe increase in the CD8+ population is significantly greater, reaching100-fold (FIG. 5). These results underscore the feasibility of usingIL-15/IL-15Rα DNA to correct defects associated with lymphopenia (e.g.,caused by total absence of IL-15 or of another etiology). Analysis oflymphocytes migrating in different organs in the presence of IL-15suggests that many acquire rapidly a memory phenotype in the absence ofantigen recognition and that IL-15 promotes re-entry of some lymphocytesinto the thymus. The issue of lymphocyte re-entry in the thymus iscontroversial, and the study of IL-15 effects may contribute to theunderstanding of this phenomenon. See, Sprent and Surh (2009) ImmunolCell Biol 87:46-49; Bosco, et al., (2009) Immunol Cell Biol 87:50-57;Agus, et al., (1991) J Exp Med 173:1039-1046. Our preliminary dataindicate that transfer of CFSE loaded thymocytes into normal miceresults in homing into the thymus only in animals receiving IL-15.

We have found that IL-15 decreases the frequency of apoptoticthymocytes, mainly by promoting their terminal differentiation intomature single positive T cells. Our results after intrathymic injectionof CFSE indicate that IL-15 increases thymic output, as reflected by thehigher frequency of fully mature CFSE labeled T cells in the spleen andlung of IL-15 treated mice.

We have further observed that the enlarged spleen size upon IL-15treatment is partially due to increased frequency of B lymphocytes,either by local proliferation, B cell migration from other compartments,or both. In addition, during in vivo experiments with adoptivetransferred CFSE-labeled splenocytes we observed IL-15-inducedproliferation of both CD4 naïve and memory T cells. In contrast to CD8+T cells, which almost universally proliferate in the presence of IL-15,the CD4+ T cell responses appear to be restricted to a subset of cells.

Example 2 Correction of Cyclophosphamide-Induced Lymphopenia byIL-15/IL-15Rα DNA Administration

Summary

The present example shows the reversal of cyclophosphamide-inducedlymphopenia in normal young mice by systemic administration of IL-15.One or two high doses of IL-15 were administered two (2) days (or two(2) and twelve (12) days) after cyclophosphamide by hydrodynamic DNAinjection. The results show that mice recover faster from lymphopeniaafter IL-15 administration in comparison to control mice withcyclophosphamide-induced lymphopenia that did not receive IL-15.Lymphocytes recovered faster in peripheral tissues after IL-15administration. NK cells were the first to recover, whereas T cellsrecovered in approximately one month. In the course of these studies, wediscovered that two administrations of IL-15 improved T cell recoveryover a single administration of IL-15. In addition, low and sustainedlevels of IL-15 provides for a more efficient repopulation oflymphocytes to the peripheral tissues in comparison to a single highdose. These results demonstrate that IL-15 is useful in treating and/orpreventing lymphopenia.

Methods

Cyclophosphamide Administration

Six-to-eight week old female Balb/c mice were obtained from CharlesRiver Laboratory (Frederick, Md.). Cyclophosphamide (Sigma) wasdissolved in pyrogen-free saline and injected intra-peritoneally (i.p.)at a dose of 200 mg/kg of body weight. Two treatments withcyclophosphamide were performed at day −4 and −2.

DNA Injection

On day 0, hydrodynamic injection of either a control vector or IL-15 andIL-15Rα expression plasmid into cyclophopshamide treated mice wasperformed. Empty vector DNA was also administered to thecyclophopshamide-untreated mice, as control. Briefly, 0.2 μg to 2 μg ofDNA in 1.6 ml of sterile 0.9% NaCl were injected into mice through thetail vein within 7 seconds using a 27.5 gauge needle. Highly purified,endotoxin-free DNA plasmids were produced using Qiagen EndoFree Giga kit(Qiagen, Hilden).

Lymphocyte Analysis

Mice were sacrificed at different time points (days 2-26) after DNAinjection and serum, bone marrow, thymus, spleen, liver and lungs werecollected for analysis.

For bone marrow lymphocyte isolation, left and right femurs werecollected and centrifuged at 13,000 for 5 min, re-suspended, andcentrifuged again (total of 3 times). Collected cells were re-suspendedin RPMI containing 10% fetal calf serum and viable cells were countedusing Acridine Orange (Molecular Probes)/Ethidium Bromide (Fisher) dye.

For splenocyte or thymocyte isolation, spleens or thymi were gentlysqueezed through a 100 μm Cell Strainer (Thomas) and washed in RPMI(Gibco) to remove any remaining lymphocytes from the organ stroma. Aftercentrifugation, the cells were re-suspended in RPMI containing 10% fetalcalf serum and counted.

To isolate lymphocytes from livers or lungs, the tissues were minced andincubated with 200 U/ml of collagenase (Sigma) and 30 U/ml of DNase(Roche) for 1 h at 37° C., then single cells were collected, centrifugedand re-suspended in complete RPMI with 10% fetal calf serum.

For phenotyping, the cells were incubated with the following mix ofdirectly conjugated anti-mouse antibodies (BD Pharmingen): CD3-APC,CD4-PerCP, CD8-PECy7, CD44-APC, CD49b-FITC, CD19-PE, CD62L-PE. Labeledcell samples were analyzed by flow cytometry using an LSR II FlowCytometer (BD) and were analyzed using FlowJo software (Tree Star, SanCarlos, Calif.).

Lymphocytes of the different group of mice were counted and compared.Statistical analyses were performed using the Prism Software Program.Comparisons of two groups were performed by non-parametric Mann-Whitneyt test. Confidence intervals were 0.05, and all p values weretwo-tailed.

Results

Two injections of cyclophosphamide at days −4 and −2 were used togenerate lymphodepleted mice. At day 0 (and also, for some mice at day10) IL-15/15Rα DNA expression vector was injected in the tail vein,which generated high systemic levels of bioactive IL-15/15Rα, aspublished (Bergamaschi, et al., J Biol Chem. (2008) 283(7):4189-99). Thebiological effects after injection of IL-15/15Rα DNA were compared tothe injection of a non-producing DNA (vector BV) as negative control incyclophosphamide-treated animals.

Different tissues, including lung, liver, spleen, thymus and bonemarrow, were extracted from mice sacrificed at days 2-26 from DNAinjection and the lymphocyte populations were studied.

Cyclophosphamide treatment had strong effects on lymphocytes, asreflected in the increased spleen weight of treated animals (FIG. 7).Four animals per time point were sacrificed and the spleen weight wasmonitored. The two groups treated with cyclophosphamide (CP+vector,treated with a non-producing DNA vector; CP+IL-15) had a smaller spleenat day 2 after DNA treatment (4 days after cyclophosphamide). At thisearly point and also at day 5 the IL-15 treated animals showed astatistically significant difference in spleen size, indicatingaccelerated recovery by IL-15.

Lung

We also analyzed lymphocyte numbers and subsets in different tissues toevaluate the effects of IL-15/15Rα administration. These experimentswere performed after one or two IL-15/15Rα DNA administrations (at days0 and 10).

Lung lymphocytes were evaluated in order to determine the effects ofIL-15/15Rα on a peripheral site, where lymphocytes need to function.IL-15 is known to affect strongly CD8+ T cells and NK cells. High levelsof IL-15 (achieved with two injections of 2 μg DNA at days 0 and 10),favors lymphocyte recovery in the lung after Cyp treatment.

Effects on Natural Killer (NK) Cells:

Mice were treated at days −4 and −2 and injected with DNA at day 0. Twogroups of mice were injected with either BV negative control DNA or withIL-15/IL-15Rα DNA. The IL-15/IL-15Rα-treated animals had a trend forhigher NK numbers for all time points. At day 14, comparison of thegroup receiving empty vector with the group of 2×IL-15/IL-15Rαadministration (DNA injections at days 0 and 10) showed that IL-15/15Rαsignificantly increased lung NK cell recovery (p=0.03).

The lymphocyte population that recovers first is the NK cells. In ourexperiments after cyclophosphamide treatment the NK cells recoveredpartially in the absence of any other intervention. IL-15/15Rαadministration accelerated this recovery. The best recovery was observedafter two IL-15 injections at days 0 and 10. Examination at day 14showed a significant increase in NK by IL-15 compared to Cyp (p=0.03).See, FIG. 8.

Effects on Lung T Cells

In contrast to NK cells, lung T cells do not recover as fast. The micewere treated and analyzed as above. Lung T cells were enumerated at day14 after the first DNA injection. It was found that total T cellsincreased at day 14 after two IL-15/Rα administrations at days 0 and 10,compared to the Cyp treated animals. See, FIG. 9.

The lung T cells were also distinguished according to expression of CD4or CD8 and compared among different groups of mice. It was found thatthe CD8+ T cells increased preferentially after IL-15/15Rαadministration at day 14 (p=0.0357). Moreover, at days 6 and 14 theCD8/CD4 ratio was increased, demonstrating the preferential stimulationof CD8+ T cells by IL-15. The ratio returns to normal by day 26, in thegroup that received IL-15/15Rα. See, FIGS. 10 and 11.

Spleen

In the spleen, we also found that T cells recover faster after twoinjections of IL-15/15Rα (p=0.0357). Similar to the results in the lung,two doses of IL-15/15Rα (days 0 and 10) were able to increase spleenlymphocytes after Cyp (p=0.03). See, FIG. 12.

Bone Marrow

Sustained high level of IL-15 (achieved with two injections of 2 μg DNAat days 0 and 10) resulted in T cell recovery in bone marrow by day 14after the first DNA injection (FIG. 13). IL-15 affected both CD4 and CD8compartments. Treatment with two administrations of IL-15/15Rα resultedin high levels of bone marrow T cells at day 14 compared to Cyp treatedanimals.

Example 3 Therapeutic Effects of IL-15 on Lymphopenia in Two DifferentMouse Strains

This example also employed Black6 mice to analyze therapeutic effects ofvarious forms of IL-15 on lymphopenia. Two different mouse strains,BALB/c and Black6, were used in these experiments. Both strains showedaccelerated lymphocyte reconstitution upon treatment with IL-15/IL-15Rα.

Treatment of Lymphoablated Mice with IL-15 DNA

Female Balb/c or Black6 mice 6-8 weeks in age were treatedintra-peritoneally with a dose of 200 mg/kg of body weight ofcyclophosphamide (CYP, FIG. 14). Two injections of CYP were performed atday −4 and day −2. At day 0 and day 5, hydrodynamic injection of eithera control DNA or DNA expressing IL-15/IL-15sRα soluble molecule wasperformed. Control vector was also delivered in CYP-untreated mice ascontrol. Mice were sacrificed at different time points: day −1 to assessthe CYP-induced lymphoablation and day 5, 10, 17 and 24 to follow immunereconstitution in presence or absence of exogenous IL-15. Differenttissues (spleen, thymus, bone marrow, lung and liver) were harvested andanalyzed for the presence of different lymphocyte subsets. Analysis wasperformed by flow cytometry after staining the cells withfluorescent-labeled antibodies.

For flow analysis, isolated cells were incubated with the followingdirectly conjugated anti-mouse antibodies (BD Pharmingen) in appropriatecombinations according to the objectives of the experiment:

-   CD3-APC or CD3-APC-Cy7, CD4-PerCp, CD8-Pacific Blue, CD44-APC,    CD62L-PE, CD19-APC-Cy7 or CD19-PeCy7, CD49b-FITC, CD25-APC-Cy7,    CD122-PE. T cells were defined as CD3⁺ cells in the lymphocyte gate;    NK cells were defined as CD3⁻CD49b⁺ cells.

For identification of Treg population (T CD4⁺CD25⁺FoxP3⁺ cells), thecells were fixed and permeabilized (eBioscience), and incubated withanti-mouse FoxP3-PeCy7 antibody (eBioscience). T effector cells weredefined as CD3⁺FoxP3⁻ lymphocytes. Therefore, the term “Teffector” asused in here refers to all T cells except Treg.

FIG. 15 shows the reconstitution of NK cell compartment in spleen andlung after CYP treatment. CYP-untreated mice were used as baselinecontrol (squares). Two injections of CYP resulted in a drastic reductionof the absolute number of NK cells in both spleen and lung (day −1). NKcells spontaneously recover between day 10 and day 14 days after controlDNA injection (triangles). One single administration of IL-15/IL-15sRαDNA was able to promote a full recovery of NK within 5 days after DNAinjection. The second IL-15/IL-15sRα expressing DNA injection resultedin an even further expansion of NK cells in both spleen and lung(circles).

FIG. 16 shows the reconstitution of T cell compartment in spleen andlung after CYP treatment. CYP-untreated mice were used as baselinecontrol (squares). Two injections of CYP resulted in a 4 fold reductionin the level of splenic T cells and in 10 fold reduction in the level ofT cells residing in the lung (day −1). The spontaneous recovery of Tcells appeared to be slower in comparison with the recovery of NK cellsand was still incomplete at day 24 after control DNA injection. Thekinetics of spontaneous recovery of T CD8 and T CD4 was similar in bothspleen and lung (triangles). Two injections of DNA expressingIL-15/IL-15sRα were able to fully reconstitute the T cell numbers within10 days after DNA administration in both spleen and lung. IL-15 promotedmainly the expansion of T CD8 cells that reached normal level at day 5after DNA injection and were boosted over normal level at day 10 afterDNA injection. IL-15 did not significantly affect the recovery of T CD4and B cells.

In addition, T cells recovering in the presence of high level ofIL-15/IL-15sRα show increased T effector (Teff)/T regulatory (Treg)ratio and increased ability to secrete IFNgamma and greaterdegranulation after in vitro stimulation. FIG. 17 is an analysis of theTeff/Treg ratio after CYP treatment for lymphodepletion and during therecovery phase. The Teff/Treg ratio increased significantly at day 10after IL-15/15sRα DNA injection.

Example 4 DNA Delivery for IL-15 to Treat Lymphopenia

In these examples, three preferred DNA vector combinations are evaluatedfor the therapeutic delivery of IL-15 to treat lymphopenia:

-   1 Co-delivery in the same cells, using preferably optimized    expression plasmids expressing IL-15 and essentially full-length    IL-15Rα, such as SEQ ID NO:13 and SEQ ID NO:14.-   2 Co-delivery in the same cells, using preferably optimized    expression plasmids expressing IL-15 and soluble (s) IL-15Rα, such    as SEQ ID NO:15.-   3 Co-delivery in the same cells, using preferably optimized    expression plasmids expressing IL-15 and IL-15Rα fusions to the    constant region of an immunoglobulin molecule (Fc) such as SEQ ID    NO:16 and SEQ ID NO:19. The construction of Fc fusion proteins is    known in the art. Such constructs have been used in vivo experiments    in mice to show that IL-15 and IL15Rα-Fc fusion heterodimers are    active in vivo.

Delivery of IL-15/IL-15Rα heterodimer by approach (1) above leads toexpression of both plasma membrane-bound and secreted IL-15/IL-15Rα.Delivery by approach (2) leads to exclusively secreted IL-15/IL-15Rαheterodimer. Delivery by approach (3) leads to a secreted bioactiveheterodimer, which is then bound to cells expressing the Fc Ab receptoron their surface. These cells can present the IL-15/IL-15RαFcheterodimer to neighboring cells, resulting in activation.

The three types of vectors have been tested in mice and have been shownto produce systemically bioactive levels of IL-15/IL-15Rα (see FIG. 18,showing expression of the three types of complexes). Because thelocalization, trafficking and stability of the different types ofcomplexes vary, the biological effects on lymphocytes is also variable.FIG. 18 shows expression of different IL-15/IL-15Rα heterodimeric formsin mice by hydrodynamic injection of DNA vectors. Mice were injected atthe tail vein (hydrodynamic delivery) with 0.1 μg of DNA expressing thedifferent forms of IL-15/IL-15Rα. Plasma levels of IL-15 were measuredat days 1 and 2.5 by R&D Quantiglo ELISA. Measurement of plasma levelsof IL-15 produced by the different vectors showed that the highestplasma levels were achieved by the DNA vector producing IL-15/IL-15RαFcfusion. The stability of the produced proteins was also different, withthe IL-15/IL-15RαFc and the IL-15/IL-15Rα full length showing thegreatest stability. The IL-15/sIL-15Rα that is not cell associated wasless stable.

Table 2 shows the CD4/CD8 ratios measured in the spleen and lung of micetreated with different IL-15/IL-15Rα heterodimeric forms, 2½ days afterhydrodynamic injection of 0.1 μg of DNA vector (see FIG. 17).

VECTOR Spleen Lung IL-15/IL-15Rα (full length) 1.36 0.8 IL-15/sIL-15Rα(soluble) 0.81 0.24 IL-15/IL-15RαFc fusion to Fc 0.63 0.52 DNA vectorcontrol 2 1.61

In these experiments, it was discovered that the different moleculeshave differential effects on lymphocytes. Therefore, the different IL-15complexes can be used alone or in combinations for the most beneficialtreatment under specific conditions. For example, delivery ofcombinations of IL-15/sRα soluble complex and IL-15/15RαFc fusioncomplex provides the opportunity to deliver both soluble and cell-boundIL-15 (through the Fc receptor) at different levels and proportions.

In addition to the different ratios of CD4/CD8 cells (as shown in Table1), the different IL-15 heterodimers also showed differences in theeffects on other surface markers of lymphocytes. FIG. 19 shows thatIL-15/15RαFc expression induced high levels of CD25 (IL-2 Receptoralpha) on both T CD4 and T CD8 cells, whereas the other forms ofIL-15/IL-15Rα heterodimers did not affect CD25 expression strongly.

FIG. 20 shows that IL-15/IL-15RαFc increased the levels of CD62L on thesurface of spleen T cells, whereas the other forms of IL-15/IL-15Rαeither did not affect or decreased average levels of CD62L on spleen Tcells. In contrast, IL-15/IL-15RαFc was less effective in increasingCD44 on spleen T cells compared to either IL-15/IL-15Rα full-length orIL-15/IL-15sRα (FIG. 21).

Example 5 Protein Delivery

As an alternative method to provide IL-15, delivery of purified proteincan be used. Protein purification from cell lines over-producingIL-15/IL-15Rα complexes has been achieved. Similar to DNA, differentforms of the heterodimer can be used alone or in combinations forobtaining the appropriate effects:

-   1 Delivery of purified IL-15/soluble (s) IL-15Rα, such as SEQ ID    NO:10 and SEQ ID NO:12.-   2 Delivery of purified IL-15/IL-15Rα Fc fusion protein (fusion to    the constant region of an immunoglobulin molecule, such as SEQ ID    NO:17 and SEQ ID NO:20)

IL-15/sIL-15Rα was purified from overproducing human 293 cells anddelivered into lympho-ablated mice. The results showed that thisheterodimer is bioactive and that it promoted the proliferation ofadoptively transferred lymphocytes (T cells, NK cells, but not B cells).

Experimental procedure (FIG. 22): Mice were treated withCyclophosphamide (Cyp) and two days later they were given 3 μg ofHPLC-purified IL-15/s15Rα protein intraperitoneally for 6 days.Splenocytes were purified from young Bl/6 mice, labeled with CFSE, and10⁷ cells were injected by the IV route to the lympho-ablated animals.Proliferation of the adoptively transferred cells was followed by CFSEdilution.

Thus, these results indicate that different forms of IL-15/IL-15Rαheterodimer have different stability, interactions in the body,processing and stability. This offers the opportunity to exploit suchproperties for using these cytokines to provide maximal benefit.Accordingly, the different forms can be combined in different ratios andadministration schedules. Different forms can be administered eithersimultaneously or sequentially.

IL-15Rα-Fc fusions previously employed have been used with variousdegrees of effectiveness. The studies exemplified in FIG. 23 show thatthe Fc fusion we used has greater plasma half-life compared toIL-15/s15Rα.

In the examples of sequences, described herein, the 205FC fusion (SEQ IDNO:17) contains the natural processing site generating the s15Rα fromthe membrane-bound form, whereas the 200FC fusion (SEQ ID NO:20) doesnot have an intact processing site. These are examples of Fc fusionsthat may be processed differently to generate non-cell associated formsafter cleavage between the 15Rα region and the antibody constant region.Additional molecules can be generated having processing sites forcleavage and generating both cell associated and soluble forms of thecytokine. Additional methods for cell attachment, other than the Fcregion are known in the art and can also be employed.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES OF SEQUENCES

SEQ ID NO: 1 Human wild-type IL-15 nucleic acid sequenceATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCTGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA SEQ ID NO: 2Human wild-type IL-15 amino acid sequence M R I S K P H L R S I S I Q CY L C L L L N S H F L T E A G I H V F I L G C F S A G L P K T E A N W VN V I S D L K K I E D L I Q S M H I D A T L Y T E S D V H P S C K V T AM K C F L L E L Q V I S L E S G D A S I H D T V E N L I I L A N N S L SS N G N V T E S G C K E C E E L E E K N I K E F L Q S F V H I V Q M F IN T S • SEQ ID NO: 3 Human improved IL-15 nucleic acid sequence (opt1)ATGCGGATCTCGAAGCCGCACCTGCGGTCGATATCGATCCAGTGCTACCTGTGCCTGCTCCTGAACTCGCACTTCCTCACGGAGGCCGGTATACACGTCTTCATCCTGGGCTGCTTCTCGGCGGGGCTGCCGAAGACGGAGGCGAACTGGGTGAACGTGATCTCGGACCTGAAGAAGATCGAGGACCTCATCCAGTCGATGCACATCGACGCGACGCTGTACACGGAGTCGGACGTCCACCCGTCGTGCAAGGTCACGGCGATGAAGTGCTTCCTCCTGGAGCTCCAAGTCATCTCGCTCGAGTCGGGGGACGCGTCGATCCACGACACGGTGGAGAACCTGATCATCCTGGCGAACAACTCGCTGTCGTCGAACGGGAACGTCACGGAGTCGGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTCCTGCAGTCGTTCGTGCACATCGTCCAGATGTTCATCAACACGTCGTGA SEQ ID NO: 4Human improved IL-15 nucleic acid sequence (opt2)ATGAGGATCAGCAAGCCCCACCTGAGGAGCATCAGCATCCAGTGCTACCTGTGCCTGCTGCTGAACAGCCACTTCCTGACCGAGGCCGGTATACACGTGTTCATCCTGGGCTGCTTTAGCGCCGGACTGCCCAAGACCGAGGCCAATTGGGTGAACGTGATCAGCGACCTGAAGAAGATCGAGGACCTCATCCAGAGCATGCACATCGACGCCACCCTGTACACCGAGAGCGATGTGCACCCCAGCTGTAAGGTGACCGCCATGAAGTGCTTTCTGCTGGAGCTGCAAGTGATCAGCCTGGAGAGCGGCGACGCCAGCATCCACGACACCGTGGAGAACCTGATCATCCTGGCCAACAACAGCCTGAGCAGCAACGGCAATGTGACCGAGAGCGGCTGTAAGGAGTGTGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACCAGCTGA SEQ ID NO: 5Homo sapiens interleukin 15 receptor, alpha (IL15RA), transcript variant1, mRNA - GenBank Accession No. NM_002189 1 cccagagcag cgctcgccacctccccccgg cctgggcagc gctcgcccgg ggagtccagc 61 ggtgtcctgt ggagctgccgccatggcccc gcggcgggcg cgcggctgcc ggaccctcgg 121 tctcccggcg ctgctactgctgctgctgct ccggccgccg gcgacgcggg gcatcacgtg 181 ccctcccccc atgtccgtggaacacgcaga catctgggtc aagagctaca gcttgtactc 241 cagggagcgg tacatttgtaactctggttt caagcgtaaa gccggcacgt ccagcctgac 301 ggagtgcgtg ttgaacaaggccacgaatgt cgcccactgg acaaccccca gtctcaaatg 361 cattagagac cctgccctggttcaccaaag gccagcgcca ccctccacag taacgacggc 421 aggggtgacc ccacagccagagagcctctc cccttctgga aaagagcccg cagcttcatc 481 tcccagctca aacaacacagcggccacaac agcagctatt gtcccgggct cccagctgat 541 gccttcaaaa tcaccttccacaggaaccac agagataagc agtcatgagt cctcccacgg 601 caccccctct cagacaacagccaagaactg ggaactcaca gcatccgcct cccaccagcc 661 gccaggtgtg tatccacagggccacagcga caccactgtg gctatctcca cgtccactgt 721 cctgctgtgt gggctgagcgctgtgtctct cctggcatgc tacctcaagt caaggcaaac 781 tcccccgctg gccagcgttgaaatggaagc catggaggct ctgccggtga cttgggggac 841 cagcagcaga gatgaagacttggaaaactg ctctcaccac ctatgaaact cggggaaacc 901 agcccagcta agtccggagtgaaggagcct ctctgcttta gctaaagacg actgagaaga 961 ggtgcaagga agcgggctccaggagcaagc tcaccaggcc tctcagaagt cccagcagga 1021 tctcacggac tgccgggtcggcgcctcctg cgcgagggag caggttctcc gcattcccat 1081 gggcaccacc tgcctgcctgtcgtgccttg gacccagggc ccagcttccc aggagagacc 1141 aaaggcttct gagcaggatttttatttcat tacagtgtga gctgcctgga atacatgtgg 1201 taatgaaata aaaaccctgccccgaatctt ccgtccctca tcctaacttt cagttcacag 1261 agaaaagtga catacccaaagctctctgtc aattacaagg cttctcctgg cgtgggagac 1321 gtctacaggg aagacaccagcgtttgggct tctaaccacc ctgtctccag ctgctctgca 1381 cacatggaca gggacctgggaaaggtggga gagatgctga gcccagcgaa tcctctccat 1441 tgaaggattc aggaagaagaaaactcaact cagtgccatt ttacgaatat atgcgtttat 1501 atttatactt ccttgtctattatatctata cattatatat tatttgtatt ttgacattgt 1561 accttgtata aacaaaataaaacatctatt ttcaatattt ttaaaatgca SEQ ID NO: 6 interleukin 15 receptor,alpha isoform 1 precursor [Homo sapiens] - GenBank Accession No.NP_002180 1 maprrargcr tlglpallll lllrppatrg itcpppmsve hadiwvksyslysreryicn 61 sgfkrkagts sltecvlnka tnvahwttps lkcirdpalv hqrpappstvttagvtpqpe 121 slspsgkepa asspssnnta attaaivpgs qlmpskspst gtteisshesshgtpsqtta 181 knweltasas hqppgvypqg hsdttvaist stvllcglsa vsllacylksrqtpplasve 241 meamealpvt wgtssrdedl encshhl SEQ ID NO: 7 Homo sapiensinterleukin 15 receptor, alpha (IL15RA), transcript variant 2, mRNA -GenBank Accession No. NM_172200 1 caggaattcg gcgaagtggc ggagctggggccccagcggg cgccgggggc cgcgggagcc 61 agcaggtggc gggggctgcg ctccgcccgggccagagcgc accaggcagg tgcccgcgcc 121 tccgcaccgc ggcgacacct ccgcgggcactcacccaggc cggccgctca caaccgagcg 181 cagggccgcg gagggagacc aggaaagccgaaggcggagc agctggaggc gaccagcgcc 241 gggcgaggtc aagtggatcc gagccgcagagagggctgga gagagtctgc tctccgatga 301 ctttgcccac tctcttcgca gtggggacaccggaccgagt gcacactgga ggtcccagag 361 cacgacgagc gcggaggacc gggaggctcccgggcttgcg tgggcatcac gtgccctccc 421 cccatgtccg tggaacacgc agacatctgggtcaagagct acagcttgta ctccagggag 481 cggtacattt gtaactctgg tttcaagcgtaaagccggca cgtccagcct gacggagtgc 541 gtgttgaaca aggccacgaa tgtcgcccactggacaaccc ccagtctcaa atgcattaga 601 gaccctgccc tggttcacca aaggccagcgccaccctcca cagtaacgac ggcaggggtg 661 accccacagc cagagagcct ctccccttctggaaaagagc ccgcagcttc atctcccagc 721 tcaaacaaca cagcggccac aacagcagctattgtcccgg gctcccagct gatgccttca 781 aaatcacctt ccacaggaac cacagagataagcagtcatg agtcctccca cggcaccccc 841 tctcagacaa cagccaagaa ctgggaactcacagcatccg cctcccacca gccgccaggt 901 gtgtatccac agggccacag cgacaccactgtggctatct ccacgtccac tgtcctgctg 961 tgtgggctga gcgctgtgtc tctcctggcatgctacctca agtcaaggca aactcccccg 1021 ctggccagcg ttgaaatgga agccatggaggctctgccgg tgacttgggg gaccagcagc 1081 agagatgaag acttggaaaa ctgctctcaccacctatgaa actcggggaa accagcccag 1141 ctaagtccgg agtgaaggag cctctctgctttagctaaag acgactgaga agaggtgcaa 1201 ggaagcgggc tccaggagca agctcaccaggcctctcaga agtcccagca ggatctcacg 1261 gactgccggg tcggcgcctc ctgcgcgagggagcaggttc tccgcattcc catgggcacc 1321 acctgcctgc ctgtcgtgcc ttggacccagggcccagctt cccaggagag accaaaggct 1381 tctgagcagg atttttattt cattacagtgtgagctgcct ggaatacatg tggtaatgaa 1441 ataaaaaccc tgccccgaat cttccgtccctcatcctaac tttcagttca cagagaaaag 1501 tgacataccc aaagctctct gtcaattacaaggcttctcc tggcgtggga gacgtctaca 1561 gggaagacac cagcgtttgg gcttctaaccaccctgtctc cagctgctct gcacacatgg 1621 acagggacct gggaaaggtg ggagagatgctgagcccagc gaatcctctc cattgaagga 1681 ttcaggaaga agaaaactca actcagtgccattttacgaa tatatgcgtt tatatttata 1741 cttccttgtc tattatatct atacattatatattatttgt attttgacat tgtaccttgt 1801 ataaacaaaa taaaacatct attttcaatatttttaaaat gca SEQ ID NO: 8 interleukin 15 receptor, alpha isoform 2[Homo sapiens] - GenBank Accession No. NP_751950 1 msvehadiwv ksyslysreryicnsgfkrk agtssltecv lnkatnvahw ttpslkcird 61 palvhqrpap pstvttagvtpqpeslspsg kepaasspss nntaattaai vpgsqlmpsk 121 spstgtteis shesshgtpsqttaknwelt asashqppgv ypqghsdttv aiststvllc 181 glsavsllac ylksrqtpplasvemeamea lpvtwgtssr dedlencshh l SEQ ID NO: 9 Improved humaninterleukin 15 (IL-15) receptor alpha (IL15Ra), transcript variant 1(OPT) atggccccga ggcgggcgcg aggctgccgg accctcggtc tcccggcgct gctactgctc60 ctgctgctcc ggccgccggc gacgcggggc atcacgtgcc cgccccccat gtccgtggag 120cacgcagaca tctgggtcaa gagctacagc ttgtactccc gggagcggta catctgcaac 180tcgggtttca agcggaaggc cggcacgtcc agcctgacgg agtgcgtgtt gaacaaggcc 240acgaatgtcg cccactggac gaccccctcg ctcaagtgca tccgcgaccc ggccctggtt 300caccagcggc ccgcgccacc ctccaccgta acgacggcgg gggtgacccc gcagccggag 360agcctctccc cgtcgggaaa ggagcccgcc gcgtcgtcgc ccagctcgaa caacacggcg 420gccacaactg cagcgatcgt cccgggctcc cagctgatgc cgtcgaagtc gccgtccacg 480ggaaccacgg agatcagcag tcatgagtcc tcccacggca ccccctcgca aacgacggcc 540aagaactggg aactcacggc gtccgcctcc caccagccgc cgggggtgta tccgcaaggc 600cacagcgaca ccacggtggc gatctccacg tccacggtcc tgctgtgtgg gctgagcgcg 660gtgtcgctcc tggcgtgcta cctcaagtcg aggcagactc ccccgctggc cagcgttgag 720atggaggcca tggaggctct gccggtgacg tgggggacca gcagcaggga tgaggacttg 780gagaactgct cgcaccacct ataatga 807 SEQ ID NO: 10 - improved humaninterleukin 15 (IL-15) receptor alpha (IL15Ra), transcript variant 1(OPT) Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala  1               5                  10                  15 Leu Leu LeuLeu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr             20                  25                  30 Cys Pro Pro ProMet Ser Val Glu His Ala Asp Ile Trp Val Lys Ser         35                  40                  45 Tyr Ser Leu Tyr SerArg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys     50                  55                  60 Arg Lys Ala Gly Thr SerSer Leu Thr Glu Cys Val Leu Asn Lys Ala 65                  70                  75                  80 Thr AsnVal Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp                 85                  90                  95 Pro Ala LeuVal His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr            100                 105                 110 Ala Gly Val ThrPro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu        115                 120                 125 Pro Ala Ala Ser SerPro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala    130                 135                 140 Ala Ile Val Pro Gly SerGln Leu Met Pro Ser Lys Ser Pro Ser Thr145                 150                 155                 160 Gly ThrThr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser                165                 170                 175 Gln Thr ThrAla Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln            180                 185                 190 Pro Pro Gly ValTyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile        195                 200                 205 Ser Thr Ser Thr ValLeu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu    210                 215                 220 Ala Cys Tyr Leu Lys SerArg Gln Thr Pro Pro Leu Ala Ser Val Glu225                 230                 235                 240 Met GluAla Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg                245                 250                 255 Asp Glu AspLeu Glu Asn Cys Ser His His Leu             260                 265 SEQID NO: 11 - improved human soluble interleukin 15 (IL-15) receptor alpha(IL-15sRa) (OPT) atggccccga ggcgggcgcg aggctgccgg accctcggtc tcccggcgctgctactgctc 60 ctgctgctcc ggccgccggc gacgcggggc atcacgtgcc cgccccccatgtccgtggag 120 cacgcagaca tctgggtcaa gagctacagc ttgtactccc gggagcggtacatctgcaac 180 tcgggtttca agcggaaggc cggcacgtcc agcctgacgg agtgcgtgttgaacaaggcc 240 acgaatgtcg cccactggac gaccccctcg ctcaagtgca tccgcgacccggccctggtt 300 caccagcggc ccgcgccacc ctccaccgta acgacggcgg gggtgaccccgcagccggag 360 agcctctccc cgtcgggaaa ggagcccgcc gcgtcgtcgc ccagctcgaacaacacggcg 420 gccacaactg cagcgatcgt cccgggctcc cagctgatgc cgtcgaagtcgccgtccacg 480 ggaaccacgg agatcagcag tcatgagtcc tcccacggca ccccctcgcaaacgacggcc 540 aagaactggg aactcacggc gtccgcctcc caccagccgc cgggggtgtatccgcaaggc 600 cacagcgaca ccacgtaatg a 621 SEQ ID NO: 12 - improvedhuman soluble interleukin 15 (IL-15) receptor alpha (IL-15sRa) (OPT) MetAla Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala  1               5                  10                  15 Leu Leu LeuLeu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr             20                  25                  30 Cys Pro Pro ProMet Ser Val Glu His Ala Asp Ile Trp Val Lys Ser         35                  40                  45 Tyr Ser Leu Tyr SerArg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys     50                  55                  60 Arg Lys Ala Gly Thr SerSer Leu Thr Glu Cys Val Leu Asn Lys Ala 65                  70                  75                  80 Thr AsnVal Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp                 85                  90                  95 Pro Ala LeuVal His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr            100                 105                 110 Ala Gly Val ThrPro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu        115                 120                 125 Pro Ala Ala Ser SerPro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala    130                 135                 140 Ala Ile Val Pro Gly SerGln Leu Met Pro Ser Lys Ser Pro Ser Thr145                 150                 155                 160 Gly ThrThr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser                165                 170                 175 Gln Thr ThrAla Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln            180                 185                 190 Pro Pro Gly ValTyr Pro Gln Gly His Ser Asp Thr Thr        195                 200                 205 SEQ ID NO: 13 Dualexpression plasmid human IL15Ra + IL15CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGCGCGCGTCGAGGAATTCGCTAGCAAGAAATGGCCCCGAGGCGGGCGCGAGGCTGCCGGACCCTCGGTCTCCCGGCGCTGCTACTGCTCCTGCTGCTCCGGCCGCCGGCGACGCGGGGCATCACGTGCCCGCCCCCCATGTCCGTGGAGCACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGGGAGCGGTACATCTGCAACTCGGGTTTCAAGCGGAAGGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACGACCCCCTCGCTCAAGTGCATCCGCGACCCGGCCCTGGTTCACCAGCGGCCCGCGCCACCCTCCACCGTAACGACGGCGGGGGTGACCCCGCAGCCGGAGAGCCTCTCCCCGTCGGGAAAGGAGCCCGCCGCGTCGTCGCCCAGCTCGAACAACACGGCGGCCACAACTGCAGCGATCGTCCCGGGCTCCCAGCTGATGCCGTCGAAGTCGCCGTCCACGGGAACCACGGAGATCAGCAGTCATGAGTCCTCCCACGGCACCCCCTCGCAAACGACGGCCAAGAACTGGGAACTCACGGCGTCCGCCTCCCACCAGCCGCCGGGGGTGTATCCGCAAGGCCACAGCGACACCACGGTGGCGATCTCCACGTCCACGGTCCTGCTGTGTGGGCTGAGCGCGGTGTCGCTCCTGGCGTGCTACCTCAAGTCGAGGCAGACTCCCCCGCTGGCCAGCGTTGAGATGGAGGCCATGGAGGCTCTGCCGGTGACGTGGGGGACCAGCAGCAGGGATGAGGACTTGGAGAACTGCTCGCACCACCTATAATGAGAATTCACGCGTGGATCTGATATCGGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGATCATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCGTCGAGGATCTGGATCCGTTAACCGATATCCGCGAATTCGGCGCGCCGGGCCCTCACGACGTGTTGATGAACATCTGGACGATGTGCACGAACGACTGCAGGAACTCCTTGATGTTCTTCTCCTCCAGCTCCTCGCACTCCTTGCAGCCCGACTCCGTGACGTTCCCGTTCGACGACAGCGAGTTGTTCGCCAGGATGATCAGGTTCTCCACCGTGTCGTGGATCGACGCGTCCCCCGACTCGAGCGAGATGACTTGGAGCTCCAGGAGGAAGCACTTCATCGCCGTGACCTTGCACGACGGGTGGACGTCCGACTCCGTGTACAGCGTCGCGTCGATGTGCATCGACTGGATGAGGTCCTCGATCTTCTTCAGGTCCGAGATCACGTTCACCCAGTTCGCCTCCGTCTTCGGCAGCCCCGCCGAGAAGCAGCCCAGGATGAAGACGTGTATACCGGCCTCCGTGAGGAAGTGCGAGTTCAGGAGCAGGCACAGGTAGCACTGGATCGATATCGACCGCAGGTGCGGCTTCGAGATCCGCATTTCTTGTCGACACTCGACAGATCCAAACGCTCCTCCGACGTCCCCAGGCAGAATGGCGGTTCCCTAAACGAGCATTGCTTATATAGACCTCCCATTAGGCACGCCTACCGCCCATTTACGTCAATGGAACGCCCATTTGCGTCATTGCCCCTCCCCATTGACGTCAATGGGGATGTACTTGGCAGCCATCGCGGGCCATTTACCGCCATTGACGTCAATGGGAGTACTGCCAATGTACCCTGGCGTACTTCCAATAGTAATGTACTTGCCAAGTTACTATTAATAGATATTGATGTACTGCCAAGTGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTGAGAACGGATATGAATGGGCAATGAGCCATCCCATTGACGTCAATGGTGGGTGGTCCTATTGACGTCAATGGGCATTGAGCCAGGCGGGCCATTTACCGTAATTGACGTCAATGGGGGAGGCGCCATATACGTCAATAGGACCGCCCATATGACGTCAATAGGAAAGACCATGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGC TATTGGSEQ ID NO: 14 Dual expression plasmid human IL15Ra + IL15tPA6CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGCGCGCGTCGAGGAATTCGCTAGCAAGAAATGGCCCCGAGGCGGGCGCGAGGCTGCCGGACCCTCGGTCTCCCGGCGCTGCTACTGCTCCTGCTGCTCCGGCCGCCGGCGACGCGGGGCATCACGTGCCCGCCCCCCATGTCCGTGGAGCACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGGGAGCGGTACATCTGCAACTCGGGTTTCAAGCGGAAGGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACGACCCCCTCGCTCAAGTGCATCCGCGACCCGGCCCTGGTTCACCAGCGGCCCGCGCCACCCTCCACCGTAACGACGGCGGGGGTGACCCCGCAGCCGGAGAGCCTCTCCCCGTCGGGAAAGGAGCCCGCCGCGTCGTCGCCCAGCTCGAACAACACGGCGGCCACAACTGCAGCGATCGTCCCGGGCTCCCAGCTGATGCCGTCGAAGTCGCCGTCCACGGGAACCACGGAGATCAGCAGTCATGAGTCCTCCCACGGCACCCCCTCGCAAACGACGGCCAAGAACTGGGAACTCACGGCGTCCGCCTCCCACCAGCCGCCGGGGGTGTATCCGCAAGGCCACAGCGACACCACGGTGGCGATCTCCACGTCCACGGTCCTGCTGTGTGGGCTGAGCGCGGTGTCGCTCCTGGCGTGCTACCTCAAGTCGAGGCAGACTCCCCCGCTGGCCAGCGTTGAGATGGAGGCCATGGAGGCTCTGCCGGTGACGTGGGGGACCAGCAGCAGGGATGAGGACTTGGAGAACTGCTCGCACCACCTATAATGAGAATTCACGCGTGGATCTGATATCGGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGATCATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCGTCGAGGATCTGGATCTGGATCCGTTAACCGATATCCGCGAATTCGGCGCGCCGGGCCCTCACGACGTGTTGATGAACATCTGGACGATGTGCACGAACGACTGCAGGAACTCCTTGATGTTCTTCTCCTCCAGCTCCTCGCACTCCTTGCAGCCCGACTCCGTGACGTTCCCGTTCGACGACAGCGAGTTGTTCGCCAGGATGATCAGGTTCTCCACCGTGTCGTGGATCGACGCGTCCCCCGACTCGAGCGAGATGACTTGGAGCTCCAGGAGGAAGCACTTCATCGCCGTGACCTTGCACGACGGGTGGACGTCCGACTCCGTGTACAGCGTCGCGTCGATGTGCATCGACTGGATGAGGTCCTCGATCTTCTTCAGGTCCGAGATCACGTTCACCCAGTTTCTGGCTCCTCTTCTGAATCGGGCATGGATTTCCTGGCTGGGCGAAACGAAGACTGCTCCACACAGCAGCAGCACACAGCAGAGCCCTCTCTTCATTGCATCCATTTCTTGTCGACAGATCCAAACGCTCCTCCGACGTCCCCAGGCAGAATGGCGGTTCCCTAAACGAGCATTGCTTATATAGACCTCCCATTAGGCACGCCTACCGCCCATTTACGTCAATGGAACGCCCATTTGCGTCATTGCCCCTCCCCATTGACGTCAATGGGGATGTACTTGGCAGCCATCGCGGGCCATTTACCGCCATTGACGTCAATGGGAGTACTGCCAATGTACCCTGGCGTACTTCCAATAGTAATGTACTTGCCAAGTTACTATTAATAGATATTGATGTACTGCCAAGTGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTGAGAACGGATATGAATGGGCAATGAGCCATCCCATTGACGTCAATGGTGGGTGGTCCTATTGACGTCAATGGGCATTGAGCCAGGCGGGCCATTTACCGTAATTGACGTCAATGGGGGAGGCGCCATATACGTCAATAGGACCGCCCATATGACGTCAATAGGAAAGACCATGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGG SEQ ID NO: 15 Dual expression plasmid humanIL15sRa(soluble) + IL15tPA6CCTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGGCGCGCGTCGAGGAATTCGCTAGCAAGAAATGGCCCCGAGGCGGGCGCGAGGCTGCCGGACCCTCGGTCTCCCGGCGCTGCTACTGCTCCTGCTGCTCCGGCCGCCGGCGACGCGGGGCATCACGTGCCCGCCCCCCATGTCCGTGGAGCACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGGGAGCGGTACATCTGCAACTCGGGTTTCAAGCGGAAGGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACGACCCCCTCGCTCAAGTGCATCCGCGACCCGGCCCTGGTTCACCAGCGGCCCGCGCCACCCTCCACCGTAACGACGGCGGGGGTGACCCCGCAGCCGGAGAGCCTCTCCCCGTCGGGAAAGGAGCCCGCCGCGTCGTCGCCCAGCTCGAACAACACGGCGGCCACAACTGCAGCGATCGTCCCGGGCTCCCAGCTGATGCCGTCGAAGTCGCCGTCCACGGGAACCACGGAGATCAGCAGTCATGAGTCCTCCCACGGCACCCCCTCGCAAACGACGGCCAAGAACTGGGAACTCACGGCGTCCGCCTCCCACCAGCCGCCGGGGGTGTATCCGCAAGGCCACAGCGACACCACGTAATGAGAATTCGCGGATATCGGTTAACGGATCCAGATCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTCCTGGGCCAGAAAGAAGCAGGCACATCCCCTTCTCTGTGACACACCCTGTCCACGCCCCTGGTTCTTAGTTCCAGCCCCACTCATAGGACACTCATAGCTCAGGAGGGCTCCGCCTTCAATCCCACCCGCTAAAGTACTTGGAGCGGTCTCTCCCTCCCTCATCAGCCCACCAAACCAAACCTAGCCTCCAAGAGTGGGAAGAAATTAAAGCAAGATAGGCTATTAAGTGCAGAGGGAGAGAAAATGCCTCCAACATGTGAGGAAGTAATGAGAGAAATCATAGAATTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGATCATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCGTCGAGGATCTGGATCTGGATCCGTTAACCGATATCCGCGAATTCGGCGCGCCGGGCCCTCACGACGTGTTGATGAACATCTGGACGATGTGCACGAACGACTGCAGGAACTCCTTGATGTTCTTCTCCTCCAGCTCCTCGCACTCCTTGCAGCCCGACTCCGTGACGTTCCCGTTCGACGACAGCGAGTTGTTCGCCAGGATGATCAGGTTCTCCACCGTGTCGTGGATCGACGCGTCCCCCGACTCGAGCGAGATGACTTGGAGCTCCAGGAGGAAGCACTTCATCGCCGTGACCTTGCACGACGGGTGGACGTCCGACTCCGTGTACAGCGTCGCGTCGATGTGCATCGACTGGATGAGGTCCTCGATCTTCTTCAGGTCCGAGATCACGTTCACCCAGTTTCTGGCTCCTCTTCTGAATCGGGCATGGATTTCCTGGCTGGGCGAAACGAAGACTGCTCCACACAGCAGCAGCACACAGCAGAGCCCTCTCTTCATTGCATCCATTTCTTGTCGACAGATCCAAACGCTCCTCCGACGTCCCCAGGCAGAATGGCGGTTCCCTAAACGAGCATTGCTTATATAGACCTCCCATTAGGCACGCCTACCGCCCATTTACGTCAATGGAACGCCCATTTGCGTCATTGCCCCTCCCCATTGACGTCAATGGGGATGTACTTGGCAGCCATCGCGGGCCATTTACCGCCATTGACGTCAATGGGAGTACTGCCAATGTACCCTGGCGTACTTCCAATAGTAATGTACTTGCCAAGTTACTATTAATAGATATTGATGTACTGCCAAGTGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTGAGAACGGATATGAATGGGCAATGAGCCATCCCATTGACGTCAATGGTGGGTGGTCCTATTGACGTCAATGGGCATTGAGCCAGGCGGGCCATTTACCGTAATTGACGTCAATGGGGGAGGCGCCATATACGTCAATAGGACCGCCCATATGACGTCAATAGGAAAGACCATGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGATTGGCTATTGG SEQ ID NO: 16--DPhuIL15sRa205FC+ huGMIL15 The capitalized, bolded region is the coding region for theIL-15Receptor alpha 205FC fusioncctggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgatggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcgggcgcgcgtcgacgctagcaagaaATGGCCCCGAGGCGGGCGCGAGGCTGCCGGACCCTCGGTCTCCCGGCGCTGCTACTGCTCCTGCTGCTCCGGCCGCCGGCGACGCGGGGCATCACGTGCCCGCCCCCCATGTCCGTGGAGCACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGGGAGCGGTACATCTGCAACTCGGGTTTCAAGCGGAAGGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACGACCCCCTCGCTCAAGTGCATCCGCGACCCGGCCCTGGTTCACCAGCGGCCCGCGCCACCCTCCACCGTAACGACGGCGGGGGTGACCCCGCAGCCGGAGAGCCTCTCCCCGTCGGGAAAGGAGCCCGCCGCGTCGTCGCCCAGCTCGAACAACACGGCGGCCACAACTGCAGCGATCGTCCCGGGCTCCCAGCTGATGCCGTCGAAGTCGCCGTCCACGGGAACCACGGAGATCAGCAGTCATGAGTCCTCCCACGGCACCCCCTCGCAAACGACGGCCAAGAACTGGGAACTCACGGCGTCCGCCTCCCACCAGCCGCCGGGGGTGTATCCGCAAGGCCACAGCGACACCACGCCGAAGTCCTGCGACAAGACGCACACGTGCCCTCCCTGCCCGGCGCCCGAGCTGCTGGGAGGTCCGAGCGTGTTCCTCTTCCCGCCCAAGCCGAAGGACACGCTCATGATCTCGCGGACTCCCGAGGTCACCTGCGTCGTGGTAGACGTCAGCCACGAGGACCCGGAGGTCAAGTTCAACTGGTACGTTGACGGCGTAGAGGTGCACAACGCGAAGACGAAGCCGCGGGAGGAGCAGTACAACTCGACGTACCGAGTCGTGTCGGTCCTGACCGTCCTGCACCAGGACTGGCTCAACGGGAAGGAGTACAAGTGCAAGGTGTCGAACAAGGCGCTCCCTGCCCCGATCGAGAAGACGATCTCGAAGGCGAAGGGCCAGCCCAGGGAGCCCCAGGTCTACACGCTCCCGCCATCGCGGGACGAGCTGACGAAGAACCAGGTTTCCCTGACGTGCCTCGTCAAGGGCTTCTACCCATCGGACATCGCGGTGGAGTGGGAGAGCAACGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCGGTGCTCGACTCGGACGGGTCGTTCTTCCTCTACTCGAAGCTGACCGTCGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCGGTGATGCACGAGGCCCTCCACAACCACTACACCCAGAAGTCGCTCAGTCTGAGCCCGGGGAAGTAATGAggatccgaattcgcggatatcggttaacggatccagatctgctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccccactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggatcatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatcgtcgaggatctggatccgttaaccgatatccgcgaattcggcgcgccgggcccTCACGACGTGTTGATGAACATCTGGACGATGTGCACGAACGACTGCAGGAACTCCTTGATGTTCTTCTCCTCCAGCTCCTCGCACTCCTTGCAGCCCGACTCCGTGACGTTCCCGTTCGACGACAGCGAGTTGTTCGCCAGGATGATCAGGTTCTCCACCGTGTCGTGGATCGACGCGTCCCCCGACTCGAGCGAGATGACTTGGAGCTCCAGGAGGAAGCACTTCATCGCCGTGACCTTGCACGACGGGTGGACGTCCGACTCCGTGTACAGCGTCGCGTCGATGTGCATCGACTGGATGAGGTCCTCGATCTTCTTCAGGTCCGAGATCACGTTCACCCAGTTCGAGATGCTGCAGGCCACCGTCCCCAGGAGTAGCAGGCTCTGGAGCCACATttcttgtcgacagatccaaacgctcctccgacgtccccaggcagaatggcggttccctaaacgagcattgcttatatagacctcccattaggcacgcctaccgcccatttacgtcaatggaacgcccatttgcgtcattgcccctccccattgacgtcaatggggatgtacttggcagccatcgcgggccatttaccgccattgacgtcaatgggagtactgccaatgtaccctggcgtacttccaatagtaatgtacttgccaagttactattaatagatattgatgtactgccaagtgggccatttaccgtcattgacgtcaatagggggcgtgagaacggatatgaatgggcaatgagccatcccattgacgtcaatggtgggtggtcctattgacgtcaatgggcattgagccaggcgggccatttaccgtaattgacgtcaatgggggaggcgccatatacgtcaataggaccgcccatatgacgtcaataggtaagaccatgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattggctattgg SEQ ID NO: 17--huIL15sRa205-Fc-underlined region isIL15sRa sequence M A P R R A R G C R T L G L P A L L L L LL L R P P A T R G I T C P P P M S V E H AD I W V K S Y S L Y S R E R Y I C N S G FK R K A G T S S L T E C V L N K A T N V AH W T T P S L K C I R D P A L V H Q R P AP P S T V T T A G V T P Q P E S L S P S GK E P A A S S P S S N N T A A T T A A I VP G S Q L M P S K S P S T G T T E I S S HE S S H G T P S Q T T A K N W E L T A S AS H Q P P G V Y P Q G H S D T T P K S C D K T H T C P P C P A P E L L GG P S V F L F P P K P K D T L M I S R T P E V T C V V V D V S H E D P EV K F N W Y V D G V E V H N A K T K P R E E Q Y N S T Y R V V S V L T VL H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P RE P Q V Y T L P P S R D E L T K N Q V S L T C L V K G F Y P S D I A V EW E S N G Q P E N N Y K T T P P V L D S D G S F F L Y S K L T V D K S RW Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K SEQ ID NO:18--huGMCSF-IL15 M W L Q S L L L L G T V A C S I S N W V N V I S D L K KI E D L I Q S M H I D A T L Y T E S D V H P S C K V T A M K C F L L E LQ V I S L E S G D A S I H D T V E N L I I L A N N S L S S N G N V T E SG C K E C E E L E E K N I K E F L Q S F V H I V Q M F I N T S SEQ ID NO:19--AG256DPhuIL15sRa200FC + huGMIL15 - The capitalized, bolded region isthe coding region for the IL-15Receptor alpha 200FC fusioncctggccattgcatacgttgtatccatatcataatatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgatggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcgggcgcgcgtcgacgctagcaagaaATGGCCCCGAGGCGGGCGCGAGGCTGCCGGACCCTCGGTCTCCCGGCGCTGCTACTGCTCCTGCTGCTCCGGCCGCCGGCGACGCGGGGCATCACGTGCCCGCCCCCCATGTCCGTGGAGCACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGGGAGCGGTACATCTGCAACTCGGGTTTCAAGCGGAAGGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACGACCCCCTCGCTCAAGTGCATCCGCGACCCGGCCCTGGTTCACCAGCGGCCCGCGCCACCCTCCACCGTAACGACGGCGGGGGTGACCCCGCAGCCGGAGAGCCTCTCCCCGTCGGGAAAGGAGCCCGCCGCGTCGTCGCCCAGCTCGAACAACACGGCGGCCACAACTGCAGCGATCGTCCCGGGCTCCCAGCTGATGCCGTCGAAGTCGCCGTCCACGGGAACCACGGAGATCAGCAGTCATGAGTCCTCCCACGGCACCCCCTCGCAAACGACGGCCAAGAACTGGGAACTCACGGCGTCCGCCTCCCACCAGCCGCCGGGGGTGTATCCGCAAGGCCCGAAGTCCTGCGACAAGACGCACACGTGCCCTCCCTGCCCGGCGCCCGAGCTGCTGGGAGGTCCGAGCGTGTTCCTCTTCCCGCCCAAGCCGAAGGACACGCTCATGATCTCGCGGACTCCCGAGGTCACCTGCGTCGTGGTAGACGTCAGCCACGAGGACCCGGAGGTCAAGTTCAACTGGTACGTTGACGGCGTAGAGGTGCACAACGCGAAGACGAAGCCGCGGGAGGAGCAGTACAACTCGACGTACCGAGTCGTGTCGGTCCTGACCGTCCTGCACCAGGACTGGCTCAACGGGAAGGAGTACAAGTGCAAGGTGTCGAACAAGGCGCTCCCTGCCCCGATCGAGAAGACGATCTCGAAGGCGAAGGGCCAGCCCAGGGAGCCCCAGGTCTACACGCTCCCGCCATCGCGGGACGAGCTGACGAAGAACCAGGTTTCCCTGACGTGCCTCGTCAAGGGCTTCTACCCATCGGACATCGCGGTGGAGTGGGAGAGCAACGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCGGTGCTCGACTCGGACGGGTCGTTCTTCCTCTACTCGAAGCTGACCGTCGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCGGTGATGCACGAGGCCCTCCACAACCACTACACCCAGAAGTCGCTCAGTCTGAGCCCGGGGAAGTAATGAggatccgaattcgcggatatcggttaacggatccagatctgctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgggtacccaggtgctgaagaattgacccggttcctcctgggccagaaagaagcaggcacatccccttctctgtgacacaccctgtccacgcccctggttcttagttccagccccactcataggacactcatagctcaggagggctccgccttcaatcccacccgctaaagtacttggagcggtctctccctccctcatcagcccaccaaaccaaacctagcctccaagagtgggaagaaattaaagcaagataggctattaagtgcagagggagagaaaatgcctccaacatgtgaggaagtaatgagagaaatcatagaatttcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactcggggggggggggcgctgaggtctgcctcgtgaagaaggtgttgctgactcataccaggcctgaatcgccccatcatccagccagaaagtgagggagccacggttgatgagagctttgttgtaggtggaccagttggtgattttgaacttttgctttgccacggaacggtctgcgttgtcgggaagatgcgtgatctgatccttcaactcagcaaaagttcgatttattcaacaaagccgccgtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggatcatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatcgtcgaggatctggatccgttaaccgatatccgcgaattcggcgcgccgggcccTCACGACGTGTTGATGAACATCTGGACGATGTGCACGAACGACTGCAGGAACTCCTTGATGTTCTTCTCCTCCAGCTCCTCGCACTCCTTGCAGCCCGACTCCGTGACGTTCCCGTTCGACGACAGCGAGTTGTTCGCCAGGATGATCAGGTTCTCCACCGTGTCGTGGATCGACGCGTCCCCCGACTCGAGCGAGATGACTTGGAGCTCCAGGAGGAAGCACTTCATCGCCGTGACCTTGCACGACGGGTGGACGTCCGACTCCGTGTACAGCGTCGCGTCGATGTGCATCGACTGGATGAGGTCCTCGATCTTCTTCAGGTCCGAGATCACGTTCACCCAGTTCGAGATGCTGCAGGCCACCGTCCCCAGGAGTAGCAGGCTCTGGAGCCACATttcttgtcgacagatccaaacgctcctccgacgtccccaggcagaatggcggttccctaaacgagcattgcttatatagacctcccattaggcacgcctaccgcccatttacgtcaatggaacgcccatttgcgtcattgcccctccccattgacgtcaatggggatgtacttggcagccatcgcgggccatttaccgccattgacgtcaatgggagtactgccaatgtaccctggcgtacttccaatagtaatgtacttgccaagttactattaatagatattgatgtactgccaagtgggccatttaccgtcattgacgtcaatagggggcgtgagaacggatatgaatgggcaatgagccatcccattgacgtcaatggtgggtggtcctattgacgtcaatgggcattgagccaggcgggccatttaccgtaattgacgtcaatgggggaggcgccatatacgtcaataggaccgcccatatgacgtcaataggtaagaccatgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcagattggctattgg SEQ ID NO: 20--huIL15sRa200-FcM A P R R A R G C R T L G L P A L L L L LL L R P P A T R G I T C P P P M S V E H AD I W V K S Y S L Y S R E R Y I C N S G FK R K A G T S S L T E C V L N K A T N V AH W T T P S L K C I R D P A L V H Q R P AP P S T V T T A G V T P Q P E S L S P S GK E P A A S S P S S N N T A A T T A A I VP G S Q L M P S K S P S T G T T E I S S HE S S H G T P S Q T T A K N W E L T A S A S H Q P P G V Y P Q G P K S CD K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S R TP E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P RE E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A LP A P I E K T I S K A K G Q P R E P Q V Y T L P P S R D E L T K N Q V SL T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D SD G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H YT Q K S L S L S P G K

What is claimed is:
 1. A composition comprising a first IL-15/IL-15Rαheterodimer comprising IL-15 bound to a soluble form of IL-15Rα, whereinthe soluble form of IL-15Rα comprises amino acids 31 to 200 of SEQ IDNO:20 and is not in the form of an Fc fusion protein; and a secondIL-15/IL-15Rα-Fc heterodimer comprising IL-15 bound to an IL-15Rα-Fcfusion protein.
 2. A composition comprising an IL-15/IL-15Rα heterodimercomprising IL-15 bound to a soluble form of IL-15Rα, wherein the solubleform of IL-15Rα comprises amino acids 31 to 200 of SEQ ID NO:20 and isnot in the form of an Fc fusion protein; and a chemotherapeutic agent.3. The composition of claim 2, wherein the chemotherapeutic agent is ananticancer agent.
 4. The composition of claim 3, wherein the anticanceragent is vinblastine, fludarabine, aclarubicin, doxorubicin, exemestane,alefacept, alemtuzumab, pamidronate, idarubicin, or cyclophosphamide.